Method of Treating Degenerative Disorders of the Nervous System

ABSTRACT

The invention herein related to methods and compositions for treating nervous system disorders. The methods comprise administration of antibodies directed towards peptides that bind to receptors important in disease progression, thus attenuating the disease.

CROSS REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/337,504 filed Dec. 27, 2011, allowed, which is a continuation of U.S.application Ser. No. 12/896,410 filed Oct. 1, 2010, abandoned, which isa divisional of U.S. application Ser. No. 12/067,792, filed Mar. 21,2008, issued as U.S. Pat. No. 7,807,645, which is a U.S. National Phaseof PCT/US2006/037211 filed Sep. 25, 2006 which claims priority from U.S.Provisional Application No. 60/720,218 filed on Sep. 23, 2005, each ofwhich is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made in part with Government support under NIH GrantNo. NS52189. The Government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 23, 2011, isnamed 24978012.txt and is 1,671 bytes in size.

FIELD

The present teachings relate to methods and compositions for treatmentof neuronal disorders.

INTRODUCTION

Degenerative neuronal disorders such as multiple sclerosis (MS) caninvolve inflammatory demyelination and autoimmune responses. Microglia,in particular perivascular microglia, are believed to be necessary notonly for the maintenance, but also for the onset of inflammatorydemyelination in central nervous system (CNS) autoimmune disease (3).Activation of microglia contributes to both neuronal (46) andoligodendrocyte death (31) via release of cytokines and nitric oxide. InMS, inflammatory processes are associated with destruction of myelinsheaths, and can also involve axonal damage that can lead to permanentfunctional deficits, such as paralysis and loss of vision (1). Residentmicroglia are considered responsible for the effector mechanism leadingto demyelination, via their ability to phagocytose myelin and secreteproinflammatory cytokines (2). However, mechanisms of perivascularmicroglia activation in inflammatory demyelination have not beenidentified.

In MS lesions, perivascular activation of microglia colocalizes withareas of blood brain barrier (BBB) disruption (5). Magnetic resonanceimaging (MRI) studies link BBB breakdown with clinical relapse (6).Moreover, in vivo imaging studies have shown that BBB disruptionprovokes the immediate and focal activation of microglia (7).

One of the earliest events coupled to BBB disruption in MS is leakage ofthe blood protein fibrinogen in the nervous system that results inperivascular deposition of fibrin (8-11). Although fibrinogen has beenprimarily studied for its functions in blood coagulation, accumulatingevidence has identified pivotal roles for fibrinogen in inflammation(12, 13) and infection (14, 15). Fibrinogen is a classic acute-phasereactant, characterized by a unique molecular structure with bindingsites for cellular receptors that regulate the inflammatory process (16,17). Research in MS animal models such as Experimental AllergicEncephalitis (EAE) in mice shows that prophylactic fibrin depletioneither by genetic depletion of fibrinogen (18) or by prophylacticadministration of anti-coagulants (18, 19) ameliorates diseasepathogenesis. However, the use of anticoagulants are potentially limitedin therapeutic value, due to the hemorrhagic side effects offibrin-depleting agents. Fibrinogen is not present in the healthy CNS,but only leaks in the brain after BBB disruption, thus serving as anenvironmental “danger” signal (4).

Recent evidence has shown that paralysis of CD11b-positive microgliaameliorates inflammatory demyelination in the presence of peripheralT-cells and macrophages (3). CD11b is the alpha chain of the CD11b/CD18integrin receptor (other names: Mac-1, α_(M)β₂, Complement Receptor 3)that in inflammatory demyelination regulates phagocytosis of myelin (21,22). Myelin phagocytosis is thought to be subjected to modulationbetween inactive and active states of the Mac-1 receptor (23).Immobilized fibrinogen and insoluble fibrin, but not soluble fibrinogen,have been identified as physiological, high-affinity ligands for Mac-1(15, 24, 25). Interestingly, in MS lesions fibrin deposition colocalizeswith areas of activated microglia (11).

SUMMARY

The present inventors have developed methods for treating degenerativedisorders of the nervous system. These methods comprise administering toa mammalian subject such as a human patient in need of treatment, atherapeutically effective amount of a composition comprising a peptideconsisting essentially of about 19 amino acids and having at least about90% sequence identity with the peptide sequencetyr-ser-met-lys-lys-thr-thr-met-lys-Ile-ile-pro-phe-asn-arg-leu-thr-Ile-gly(YSMKKTTMKIIPFNRLTIG) (SEQ ID NO: 1).

Some aspects of the invention include compositions for the treatment ofa degenerative disorder of the nervous system. The compositions comprisea peptide consisting essentially of about 19 amino acids and having atleast about 90% sequence identity with the amino acid sequenceYSMKKTTMKIIPFNRLTIG (SEQ ID NO: 1), and a pharmaceutically acceptableexcipient.

In other aspects, the invention includes use of a peptide in themanufacture of a medicament for the treatment of a neurodegenerativedisease, wherein the peptide consists essentially of about 19 aminoacids and having at least about 90% sequence identity with the peptidesequence YSMKKTTMKIIPFNRLTIG (SEQ ID NO: 1).

In yet other aspects, the invention includes methods of inhibitingmicroglia activation. These methods comprise contacting the microgliawith a composition comprising a peptide consisting essentially of about19 amino acids and having at least about 90% sequence identity with thepeptide sequence YSMKKTTMKIIPFNRLTIG (SEQ ID NO: 1).

In yet other aspects, the invention includes methods of preventingdevelopment of a neurological disorder. These methods compriseadministering to a subject an effective amount of a compositioncomprising a peptide consisting essentially of about 19 amino acids andhaving at least about 90% sequence identity with the peptide sequencetyr-ser-met-lys-lys-thr-met-lys-ile-ile-pro-phe-asn-arg-leu-thr-ile-glyYS MKKTTMKIIPFNRLTIG) (SEQ ID NO: 1).

In various configurations, the microglia can be microglia comprised by amammalian subject, and the peptide can be comprised by a compositionfurther comprising a pharmaceutically acceptable excipient.

In the various aspects and configurations of the invention, the peptidecan consist essentially of the amino acid sequence YSMKKTTMKIIPFNRLTIG(SEQ ID NO: 1), or the peptide can consist of the sequenceYSMKKTTMKIIPFNRLTIG (SEQ ID NO: 1).

In various aspects and configurations of the invention, the disease ordisorder can be multiple sclerosis, spinal cord injury, stroke, orAlzheimer's Disease.

In one aspect, the peptide can be an isolated polypeptide comprising anamino acid sequence having at least about 45%, 46%, 47%, 48%, 49%, 50%,51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%,65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity toYSMKKTTMKIIPFNRLTIG (SEQ ID NO: 1).

These and other features, aspects and advantages of the presentteachings will become better understood with reference to the followingdescription, examples and appended claims.

DRAWINGS

Those of skill in the art will understand that the drawings, describedbelow, are for illustrative purposes only. The drawings are not intendedto limit the scope of the present teachings in any way.

FIG. 1 illustrates IsoB4/DAPI immunostained microglia cultured onimmobilized fibrinogen have increased cell body size adopting anamoeboid morphology (right column). Untreated primary microglia showsmall cell bodies and thin, bipolar processes (left column). LPS-treatedcells show activated morphology characterized by cell body swelling(middle column). Top row shows antibody staining; middle row shows DAPIstaining of nuclei; bottom row shows combined antibody and DAPIstaining. Scale bar, 26 μm; inset, 21 μm.

FIG. 2 illustrates Quantification of microglia activation. The datareveals a dramatic increase upon fibrinogen stimulation.

FIG. 3 depicts fibrinogen stimulated microglia showed increasedphagocytosis of fluorescent E. coli as compared to untreated microglia.LPS served as a positive control.

FIG. 4 depicts deconvolution microscopy of primary microglia revealedsignificant rearrangements of the cyto skeleton upon treatment withfibrinogen. Micro glia were stained with antibodies to actin andβ-tubulin (top row) and the nucleus stained with DAPI (middle row).Bottom row shows combined antibody and DAPI staining. Scale bar, 4.4 μm.

FIG. 5 depicts fibrinogen-induced morphologic activation of microgliablocked by the addition of a rat anti-CD11b neutralizing antibody(M1/70). Rat IgG (control) did not change the effects of fibrinogen inmicroglia activation. Top row shows antibody staining; middle row showsDAPI staining of nuclei; bottom row shows combined antibody and DAPIstaining. Scale bar, 39 μm; inset, 17 μm.

FIG. 6 depicts quantitation of microglia activation revealing that theMac-1 neutralizing antibody blocks fibrinogen-induced activation but notLPS activation of microglia.

FIG. 7 depicts increased microglia phagocytosis upon fibrinogenstimulation blocked by the addition of a PI3K inhibitor (LY294002) anddiminished in the presence of a CD11b neutralizing antibody. A controlanti-TLR4 or IgG showed no reduction in fibrinogen stimulatedphagocytosis.

FIG. 8 depicts Western blots showing increased active RhoA and Aktactivation upon fibrinogen stimulation of microglia. In both assays, LPSserved as a positive control.

FIG. 9 depicts confocal microscopy demonstrating spatial interactionbetween CD11b-positive cells (top right) and fibrin (top left), DAPIstaining (bottom left) and the combination (bottom right) in spinalcords from PLP139-151 immunized mice.

FIG. 10 depicts confocal double immunofluorescence showing fibrin(middle row) surrounding an activated microglia (top row) in a humanearly demyelinating plaque of acute MS. Bottom row shows combinedantibody and DAPI staining. Scale bars, 6.7 μm, E; 15 μm, F.

FIG. 11 depicts immunofluorescence of control (left panel) andfibrin-depleted (right panel) spinal cord with antibodies againstfibrin(ogen) (top row) and CD11b (middle row). Activated CD11b positivecells colocalize with fibrin deposition in the control spinal cord(bottom row). Scale bar, 8.4 μm

FIG. 12 depicts clinical scores of PLP139-151 immunized mice. Fibrindepletion begins after the first paralytic episode. Fibrin depleted mice(bottom curve, n=8) did not develop 1st or 2nd relapses as compared tocontrol mice (upper curve, n=10).

FIG. 13 depicts the results of a rotarod motor strength and coordinationtest. Fibrin-depleted mice outperformed control mice on day 25postimmunization. Data are represented as the mean clinical score andare mean±SEM.

FIG. 14 depicts clinical scores of MOG35-55 immunized mice.Fibγ^(390-396A) mice (triangles, n=17) show statistically significantlower clinical scores (*P<0.05, **P<0.01) than Fibγ^(WT) control mice(squares, n=15).

FIG. 15 depicts individual clinical scores from Fibγ^(390-396A) andFibγ^(WT) mice on days 17 and 31 post-immunization.

FIG. 16 depicts histological analysis of spinal cord sections revealedincreased inflammation in the FibγWT mice versus the Fibγ^(390-396A)mice.

FIG. 17 depicts a survival curve of Fibγ^(390-396A) and Fibγ^(WT) miceafter MOG35-55 immunization. Fibγ^(390-396A) (upper curve) had a greatersurvival rate than the Fibγ^(WT) mice (lower curve).

FIG. 18 depicts rotarod analysis of Fibγ_(390-396A) and Fibγ^(WT) miceon day 13. Fibγ^(390-396A) significantly outperformed their wild-typecounterparts in a behavioral test designed to assess motor skillfunction. (n=7 mice per group)

FIG. 19 depicts increased activation IsoB4 positive cells in Fibγ^(WT)mice as compared to Fibγ^(390-396A) mice. Scale bar, 58 μm. *, P<0.05;**, P<0.01. Data are represented as the mean clinical score and aremean±SEM.

FIG. 20 depicts microglia activation upon fibrinogen stimulation isattenuated in the presence of γ³⁷⁷⁻³⁹⁵. Top row shows antibody staining;middle row shows DAPI staining of nuclei; bottom row shows combinedantibody and DAPI staining. Scale bar, 33 μm; inset, 29 μm.

FIG. 21 depicts the quantitation of microglia activation reveals thatγ³⁷⁷⁻³⁹⁵ diminishes fibrinogen stimulated microglia activation but hasno effect on LPS activation.

FIG. 22 shows that treatment of microglia with γ³⁷⁷⁻³⁹⁵ blocksfibrinogen-induced Akt activation in vitro. LPS activation of Akt isunaffected by γ³⁷⁷⁻³⁹⁵ treatment.

FIG. 23 depicts clinical scores from γ³⁷⁷⁻³⁹⁵ peptide vaccinated mice.Mice were immunized with γ³⁷⁷⁻³⁹⁵ peptide preceding EAE induction withPLP139-151 peptide. Vaccinated mice (triangles, n=15) had significantlyreduced clinical scores, as compared to control mice (squares, n=15).

FIG. 24 depicts clinical scores from PLP139-151 immunized mice where theγ³⁷⁷⁻³⁹⁵ peptide was administered every day intranasally after the peakof the initial paralytic episode. γ³⁷⁷⁻³⁹⁵ treated mice (triangles,n=14) did not show a relapse at approximately day 30 as compared to thecontrols (squares, n=13). *, P<0.05; ** P<0.01. Data are represented asthe mean clinical score and are mean±SEM. Immunostaining for Mac-3 andiNOS on day 30 after immunization shows dramatic reduction of microgliaactivation in the γ³⁷⁷⁻³⁹⁵ peptide-treated mice after EAE induction(FIG. 25), when compared to control (FIG. 26). Scale bar, 20 μm; Inset10 μm.

FIG. 25 shows dramatic reduction of microglia activation in the γ³⁷⁷⁻³⁹⁵peptide-treated mice after EAE induction when compared to FIG. 24. Scalebar, 20 μm; Inset 10 μm.

FIG. 26 shows a control for FIGS. 24 and 25. Scale bar, 20 μm; Inset 10μM.

FIG. 27 depicts the quantitation showing a 2-fold reduction in Mac-3, a4.2-fold reduction in iNOS after γ³⁷⁷⁻³⁹⁵ peptide treatment, while thereare no major differences in T cell infiltration.

FIG. 28 depicts in vivo clotting time assayed in the presence of 30 μgof γ³⁷⁷⁻³⁹⁵ peptide, the dose administered in vivo daily, and 90 μg ofγ³⁷⁷⁻³⁹⁵ peptide. Neither dose affected blood clotting times.

FIG. 29 depicts prothrombin times assayed in the presence of 30 μg ofγ³⁷⁷⁻³⁹⁵ peptide, the dose administered in vivo daily, and 90 μg ofγ³⁷⁷⁻³⁹⁵ peptide. Neither dose affected blood clotting times.

FIG. 30 depicts In vitro fibrin polymerization examined in the presenceof the γ³⁷⁷⁻³⁹⁵ peptide. GPRP, a known inhibitor of fibrin formation,significantly attenuated fibrin formation while the γ³⁷⁷⁻³⁹⁵ peptide hadno effect on fibrin polymerization.

FIG. 31 illustrates a model for the role of fibrin-induced activation ofmicroglia in inflammatory demyelination. BBB disruption permits theleakage of fibrinogen, the high affinity ligand for Mac-1, in the CNSparenchyma. Fibrinogen is converted to fibrin and functions as thespatial signal to induce local activation of microglia via activation ofthe Mac-1 integrin receptor and induction of signaling pathwaysdownstream of Mac-1, such as Akt and Rho resulting in phagocytosis thatcould determine the extent of tissue damage in inflammatorydemyelination.

FIG. 32 depicts fibrin depletion decreasing in microglia activation inEAE. Spinal cord sections from mice immunized with PLP₁₃₉₋₁₅₁ peptideshow increased CD11b-positive reactivity and fibrin deposits (middlecolumn) as compared to non-immunized controls (left column) orfibrin-depleted mice (right column). Scale bar, 39 μm.

FIG. 33 depicts fibrin depletion reducing demyelinating lesions in EAE.Luxol Fast Blue/Nuclear Red staining of control cerebellum showsinflammation (asterisk) and demyelination (white area, arrow).Fibrin-depleted mice show normal cerebellar morphology. Staining ofcontrol spinal cord sections show large areas of demyelination (arrows)while fibrin-depleted spinal cord sections show normal myelinmorphology. Scale bar, 42 μm (spinal cord), 105 μm (cerebellum).

FIG. 34 depicts a splenocyte proliferation assay. Splenocytes fromcontrol and fibrin depleted mice subjected to PLP₁₃₉₋₁₅₁ EAE revealed nodifference in proliferation under untreated or antigen stimulatedconditions (PLP₁₃₉₋₁₅₁).

FIG. 35 depicts flow cytometry analysis on splenocytes from control orγ³⁷⁷⁻³⁹⁵ peptide treated mice. Splenocytes were harvested from miceimmunized with PLP₁₃₉₋₁₅₁ at the peak of the first relapse andimmunostained with six markers of the peripheral immune response. Theγ³⁷⁷⁻³⁹⁵ peptide treatment had no significant effect on peripheralimmune cells compared to control. (A) Untreated CD4 T cells; (B)γ³⁷⁷⁻³⁹⁵ treated CD4 T cells; (C) Untreated CD8 T cells; (D) γ³⁷⁷⁻³⁹⁵treated CD8 T cells; (E) Untreated CD11b macrophages; (F) γ³⁷⁷⁻³⁹⁵treated CD11b macrophages; (G) Untreated CD11c dendritic cells; (H)γ³⁷⁷⁻³⁹⁵ treated CD11c dendritic cells; (I) Untreated CD19 B cells; (J)γ³⁷⁷⁻³⁹⁵ treated CD19 B cells; (K) Untreated B220 B cells; and (L)γ³⁷⁷⁻³⁹⁵ treated B220 B cells.

DETAILED DESCRIPTION

The methods and compositions described herein utilize laboratorytechniques well known to skilled artisans and can be found in laboratorymanuals such as Sambrook, J., et al., Molecular Cloning: A LaboratoryManual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 2001; Spector, D. L. et al., Cells: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1998; andHarlow, E., Using Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1999. Pharmaceutical methodsand compositions described herein, including methods for determinationof therapeutically effective amounts, and terminology used to describesuch methods and compositions, are well known to skilled artisans andcan be adapted from standard references such as Remington: the Scienceand Practice of Pharmacy (Alfonso R. Gennaro ed. 19th ed. 1995);Hardman, J. G., et al., Goodman & Gilman's The Pharmacological Basis ofTherapeutics, Ninth Edition, McGraw-Hill, 1996; and Rowe, R. C., et al.,Handbook of Pharmaceutical Excipients, Fourth Edition, PharmaceuticalPress, 2003.

As used herein, the term “peptide” includes polypeptides consisting of,or consisting essentially of, a polypeptide chain of at least 10 aminoacids up to about 30 amino acids, and salts thereof. A peptide cancomprise a modified backbone and individual residue analogs.

The present inventors have found that the fibrin gamma 377-395(γ³⁷⁷⁻³⁹⁵) peptide,tyr-ser-met-lys-lys-thr-thr-met-lys-ile-ile-pro-phe-asn-arg-leu-thr-11e-gly(YSMKKTTMKIIPFNRLTIG) (SEQ ID NO: 1) can block microglia activation invitro, and is also able to reverse relapsing paralysis in an animalmodel of MS. This is the first demonstration of a therapy to reducemicroglia activation. This treatment can lead to great improvements inpatient health and quality of life, not only for MS therapy, but alsofor other neurodegenerative diseases with blood-brain barrier disruptionor vascular injury such as spinal cord injury, stroke and Alzheimer'sDisease.

Multiple Sclerosis (MS) is a chronic inflammatory demyelinating diseaseof the nervous system in which an inflammatory process is associatedwith destruction of myelin sheaths and later with axonal damage leadingto permanent functional deficits, such as paralysis, and loss of vision(1). Resident microglia are considered responsible for the effectormechanism leading to demyelination via their ability to phagocytosemyelin and secrete proinflammatory cytokines (2). Microglia arenecessary not only for the maintenance, but also for the onset ofinflammatory demyelination in central nervous system (CNS) autoimmunedisease (3). The mechanisms of perivascular microglia activation ininflammatory demyelination as well as a strategy to limit theiractivation in MS has not been identified. This invention is not limitedto MS, and teaches more broadly a method of treating nervous systemdisorders. MS is but one example of the diseases and conditions that canbe treated using the novel compositions and methods taught herein. Oneskilled in the art can use this disclosure to treat the broad class ofnervous system disorders. These include, but are not limited to,multiple sclerosis, spinal cord injury, stroke and Alzheimer's Disease.

The present inventor shows that inhibition of the inflammatory but notthe coagulative properties of fibrinogen is sufficient to suppressclinical symptoms and disease pathogenesis in an animal model of MS. Theinventor further shows that fibrinogen/Mac-1 interaction induces signaltransduction pathways to activate microglia and has identified theγ³⁷⁷⁻³⁹⁵ fibrin epitope (36) as a therapeutic target for MS. The resultsidentify fibrinogen as a microglial activation signal. Without beinglimited by theory, the study suggests the following model for the roleof fibrin in inflammatory demyelination (FIG. 31). 1) BBB disruption,which precedes lesion development and clinical symptoms in MS (6 45),permits the leakage of fibrinogen perivascularly in the brain. 2)Fibrinogen is converted to fibrin either by pro-coagulant factorsproduced in the nervous system or by factors from the blood that enterthe brain together with fibrinogen. 3) Fibrinogen conversion to fibrinallows the exposure of the cryptic fibrinogen epitope γ³⁷⁷⁻³⁹⁵ thusallowing interactions of fibrinogen with its Mac-1 integrin receptor. 4)Fibrin/Mac-1 interactions induce local activation of microglia. 5)Fibrin induces activation of Akt and Rho signaling in microgliaresulting in cytoskeletal rearrangement leading to increased phagocyticcapacity. 6) Fibrin-mediated microglia phagocytosis could determine theextent of tissue damage in MS. Given the colocalization of inflammatorydemyelinating lesions in MS with fibrin deposition, the data suggestthat fibrin/Mac-1 in the CNS parenchyma provides a spatial signal formicroglia activation and determines the area of demyelination in MS. Theinventor thus sought to design a therapeutic strategy that would blockthe damaging effects of fibrin in the nervous system without affectingits beneficial effects in blood coagulation by identifying and targetingthe fibrin receptor in the nervous system, and determined that theγ³⁷⁷⁻³⁹⁵ peptide of fibrin induces a reduction of iNOS-positivemicroglia, suggesting that inhibition of fibrin/Mac-1 interactionreduces microglia activation that in MS could mediate secondary damagingeffects on other cell types of the nervous system.

We sought to design a therapeutic strategy that would block the damagingeffects of fibrin in the nervous system without affecting its beneficialeffects in blood coagulation by identifying and targeting the fibrinreceptor in the nervous system. Interaction of fibrin with its receptorshas been previously used for drug development. Fibrin mediates bloodcoagulation via binding to the platelet integrin receptor α_(IIb)β₃.Development of an antibody that blocks binding of fibrin to itsα_(IIb)β₃ receptor (ReoPro®, abciximab) is a successful thrombolytictreatment for atherosclerosis (20). The α_(IIb)β₃ integrin receptor isexpressed exclusively by platelets and not by cells in the nervoussystem. We hypothesized that if we identified the specific cell type inthe nervous system that is affected by fibrin and the receptor thatfibrin utilizes to mediate this effect; we would be able to inhibit thecellular functions of fibrin in the nervous system without affecting itscoagulative properties in the blood.

Recent evidence showed that paralysis of CD11b-positive microgliaameliorates inflammatory demyelination in the presence of peripheralT-cells and macrophages (3). Myelin phagocytosis is thought to besubjected to modulation between inactive and active states of the Mac-1receptor (23). Immobilized fibrinogen, insoluble fibrin but not solublefibrinogen have been identified as physiological, high-affinity ligandsfor Mac-1 (15, 24, 25). Interestingly, in MS lesions fibrin depositioncolocalizes with areas of activated microglia (11). Here we show thatfibrin serves as an environmental signal to induce the differentiationof microglia to phagocytes via the Mac-1 (CD11b/CD18) integrin receptor.Moreover, the fibrin γ³⁷⁷⁻³⁹⁵ peptide (the binding epitope of fibrin toCD11b) functions as an inhibitor of microglia activation. Finally, weuse multiple approaches, such as knock-in mice, vaccination andintranasal peptide delivery to test the efficacy of targeting the fibrinγ³⁷⁷⁻³⁹⁵ epitope as a novel therapeutic strategy for inflammatorydemyelination. Our study shows for the first time that fibrin inducesmicroglia activation and that targeting the inflammatory, but not thecoagulative properties of fibrinogen is sufficient to suppress microgliaactivation and inflammatory demyelinating disease.

In this study we show for the first time that inhibition of theinflammatory but not the coagulative properties of fibrinogen issufficient to suppress clinical symptoms and disease pathogenesis in ananimal model of MS. We show that fibrinogen/Mac-1 interaction inducessignal transduction pathways to activate microglia and have identifiedthe γ³⁷⁷⁻³⁹⁵ fibrin epitope as a therapeutic target for MS. Our resultsidentify fibrinogen as a microglial activation signal. Fibrinogen is notpresent in the healthy CNS, but only leaks in the brain after BBBdisruption, thus serving as an environmental “danger” signal (4).Moreover, fibrinogen contains a binding motif for Mac-1 that allows theinduction of Iigand/receptormediated activation of microglia.

The present study identifies fibrinogen as a ligand for Mac-1 onmicroglia cells. Complement and in particular iC3b is well establishedas a ligand for Mac-1 (28). The presence of immunoglobulins andactivated complement is restricted to the pattern II MS lesions (1).Since microglia activation and phagocytosis is a common feature for allsubtypes of MS, other factors in the demyelinating lesion couldpotentially mediate microglia activation. Since BBB disruption is acommon feature for different types of MS lesions, our study suggeststhat fibrin could serve as a broad activation signal for microglia inthe CNS. The γ³⁷⁷⁻³⁹⁵ peptide induced a reduction of iNOS-positivemicroglia, suggesting that inhibition of fibrin/Mac-1 interactionreduces microglia activation that in MS could mediate secondary damagingeffects on other cell types of the nervous system, such as neuronaldeath.

The identification of Mac-1 integrin as the fibrin receptor thatmediates microglia activation was the basis to specifically block thefunctions of fibrin in the nervous system without affecting itsphysiological functions in blood coagulation. Two previous studies havedetermined that inhibiting the coagulative properties of fibrinogen byuse of anti-coagulants such as ancrod in a prophylactic manner canameliorate symptoms of EAE (19, 47). In the present study the inventorfurther demonstrates that administration of ancrod after the onset ofparalysis can suppress EAE (FIGS. 11-13 and FIGS. 32-33). However, theuse of anti-coagulants could have limited clinical applications to achronic disease such as MS due to adverse hemorrhagic effects caused byprolonged anti-coagulant treatment. Since α_(IIb)β₃ binds fibrinogen atthe γ⁴⁰⁸⁻⁴¹¹ epitope, targeting the Mac-1 binding epitope of fibrinogeneither genetically (15), or pharmacologically (our study) does notaffect the coagulation properties of fibrinogen. Moreover, the γ³⁷⁷⁻³⁹⁵is a cryptic epitope in the fibrinogen molecule and is exposed only whenfibrinogen is immobililized or converted to fibrin (25, 36). The presentinvestigations demonstrate that inhibition of the interaction of fibrinwith Mac-1 either genetically or pharmacologically inhibits microgliaactivation and can suppresses paralysis in mice.

Fibrin/Mac-1 mediated activation of monocytes cells is well established(17). Our study shows that fibrinogen depletion or treatment with theγ³⁷⁷⁻³⁹⁵ peptide attenuates the clinical symptoms in EAE by primarilyameliorating the microglia/macrophage response without interfering withT cell activation. These results are in accordance with previous studiesthat have established that depletion of macrophages (43) or microglia(3) in EAE results in amelioration of clinical symptoms in the presenceof functional T cell activation. In addition to CD11b, fibrinogen bindsto the CD11c chain of the CD11c/CD18 integrin (48). The γ³⁷⁷⁻³⁹⁵ fibrinpeptide blocks the binding of fibrinogen to both CD11b and CD11cintegrins (36). The CD11c-positive perivascular dendritic cells areresponsible for the presentation of myelin antigens (49, 50).

To investigate how fibrinogen increased microglia phagocytosis, weexamined whether fibrinogen regulates RhoA and PI3K, the two majorsignaling pathways that mediate the cytoskeletal rearrangements for theinduction of phagocytosis (23). We first examined if fibrinogen inducedRhoA activation in microglia, since RhoA is required for type II,Mac-1-mediated phagocytosis (24). Fibrinogen induced a 1.9-fold increaseof active RhoA as assessed by an increased interaction with GST-Rhotekinbinding protein which binds only GTP-bound, or active, RhoA (FIG. 9). Incomparison, LPS stimulation resulted in a 2.4-fold activation of Rho. Wenext examined whether fibrinogen stimulation activated PI3K, sincephosphatidylinositol 3,4,5-trisphosphate (PIP3) is required for theprogression of Mac-1 phagocytosis (25). Production of the signalinglipid, PIP3, was assessed by examining a direct downstream effector, theserine/threonine kinase Akt. Fibrinogen stimulation resulted in a24-fold increase in Akt phosphorylation while LPS stimulation resultedin a 34-fold increase (FIG. 9). Furthermore, blocking the PI3K signalingpathway using LY294002, inhibited the fibrin-induced increase ofphagocytosis in microglia (FIG. 8), suggesting that PI3K is downstreamof fibrin/Mac-1 signaling in microglia cells. Overall, these datademonstrate that fibrinogen activates both RhoA and Akt, two majorpathways in microglia that redistribution of the cytoskeleton, andultimately resulting in increased phagocytosis.

Our in vitro data show that fibrin activates microglia cells. Toinvestigate whether fibrin is required for the activation of Mac-1positive cells in vivo, we examined whether fibrin depletion wouldreduce the activation of CD11b-positive cells in Experimental AutoimmuneEncephalomyelitis (EAE). We administered the anti-coagulant ancrod (26)in an established remitting-relapsing model of EAE induced by thePLP₁₃₉₋₁₅₁ peptide in SJL/J mice (27) after the development of the firstparalytic incidence. Untreated mice show dramatic activation of CD11b+cells characterized by thick processes (FIG. 11, left panel, green) 29days after immunization. By contrast, in mice treated with ancrod CD11b+cells appear with ramified morphology (FIG. 11, right panel, green)resembling CD11b+ cells that are present in the normal spinal cord ofnon-immunized mice (FIGS. 23-27). Similar to MS lesions (9), areas ofcolocalization of fibrin (red) with IsoB4 positive cells (green) areobserved in untreated mice (data not shown). Histopathology wasperformed on cerebellum and spinal cords from mice treated with ancrodas well as controls. LFB/NR staining revealed extensive inflammatorylesions in the cerebellum (FIGS. 28-30, asterisk) and the spinal cordsof untreated mice. By contrast, mice treated with ancrod did not showsigns of inflammation (FIGS. 28-30). Demyelination was observed in thecerebellum and spinal cord (FIGS. 28-30, arrows) of untreated mice,while demyelination was minimal in ancrod-treated mice. Quantificationin the cerebellum and spinal cord showed a 7-fold and a 2-fold decreaseof the demyelinated area in ancrod-treated versus untreated controlmice. Immunostaining for T cells using an anti-CD3 cell marker, did notreveal differences in the number of T cells between ancrod-treated anduntreated mice (data not shown). In addition, there was no difference inproliferation upon stimulation with PLP₁₃₉₋₁₅₁ in spleenocytes isolatedfrom untreated and ancrod-treated mice, further suggesting that CD11b+cells are the major cell target of fibrin in the nervous system.Fibrin-depleted mice recovered faster from the first paralytic incidenceand in contrast to control mice never relapsed (FIG. 12). In a rotarodbehavioral test, performed before the second relapse of the untreatedgroup, fibrin-depleted mice showed a 3-fold increase of motor strengthand coordination when compared to the untreated group (FIG. 13).Overall, these results suggest that fibrin is a major contributor to thelocal activation of CD11b+ cells in vivo. In addition, this is the firstdemonstration that anti-coagulants improve clinical symptoms and reduceinflammatory demyelination when administered after the onset ofparalytic symptoms.

To examine the contribution of fibrinogen signaling through Mac-1 ininflammatory demyelination in vivo, we subjected mice with a knock-inmutation at the C-terminus of the gamma chain of fibrinogen toMOG-induced EAE (28). This mutation of seven amino acids at residues(N³⁹⁰RLSIGE³⁹⁶) (SEQ ID NO: 3) to alanine residues (A³⁹⁰AAAAAA³⁹⁶) (SEQID NO: 4) abolishes fibrinogen binding to the Mac-1 receptor. Thesemice, termed Fib^(Mac-1), and their age- and sex-matched littermatecontrols (Fib^(wt)) were immunized with MOG peptide (FIG. 14).Fib^(Mac-1) mice showed an average clinical score of 2.2 (ataxia) at day17 after immunization, while the Fib^(wt) mice developed clinicalsymptoms of paralysis, showing an average score of 3.66 (hind limbparalysis). In the Fib^(wt) mouse group 11 out of 15 mice wereparalyzed, by contrast to 4 out of 16 of the Fib^(Mac-1) mice (clinicalscore>3) (FIG. 15), further demonstrating decreased severity of EAE. Inaddition to the clinical score, motor function was tested by rotarodanalysis. Fib^(Mac-1) mice showed a 1.5 fold increase in motor skills,when compared to Fib^(wt) mice (Fib^(Mac-1), 269±9 sec vs. Fib^(wt),169±23 sec, P<0.05) (FIG. 18). Analysis of the inflammatory index showeda three fold decrease in Fib^(Mac-1) mice, when compared to Fib^(wt)(FIG. 16). In accordance, histological analysis of spinal cord sectionsrevealed a decrease in the extent of IsoB4-positive cells in Fib^(Mac-1)mice, when compared to Fib^(wt) controls (FIG. 19). Decrease in clinicalseverity was also observed at later time points after the immunization(FIGS. 14 and 15). In the Fib^(wt) mouse group, 4 out of 15 mice were inmoribund state (clinical score 5), by contrast to 1 out of 17 of theFib^(Mac-1) mice (FIG. 17). Overall, these results suggest that theγ³⁹⁰⁻³⁹⁶ binding site of fibrinogen to Mac-1 determines the severity ofinflammatory demyelination.

Both the in vitro (FIGS. 5-10) and in vivo experiments (FIGS. 14-19)show that fibrinogen signaling through the Mac-1 receptor contributes tomicroglia activation and regulates the severity of inflammatorydemyelination in EAE. Biochemical studies identified that the γ³⁷⁷⁻³⁹⁵peptide (YSMKKTTMKIIPFNRLTIG) (SEQ ID NO: 1) blocks fibrin binding toMac-1 (29). The γ³⁷⁷⁻³⁹⁵ is a cryptic epitope in the fibrinogen moleculeand is exposed only when fibrinogen is immobilized or converted tofibrin (25, 36). We used 200 μM of peptide, a concentration shown toinhibit adhesion of Mac-1 overexpressing cells to immobilized fibrinogen(37), to examine whether the γ³⁷⁷⁻³⁹⁵ peptide could inhibit microgliaactivation. Fibrinogen treatment resulted in 71±4.9% of activatedmicroglia, when compared to 38.3±9.8% after addition of the γ³⁷⁷⁻³⁹⁵peptide (P<0.05) (FIGS. 20 and 21), suggesting that the γ³⁷⁷⁻³⁹⁵ peptidereduced fibrin-mediated microglia activation. The γ³⁷⁷⁻³⁹⁵ peptide didnot affect the activation state of untreated microglia and did notinhibit LPS-mediated microglia activation (FIG. 21), further suggestingthe specificity of γ³⁷⁷⁻³⁹⁵ to block activation of Mac-1 by fibrin. Inaccordance, examination of Akt phosphorylation showed that the γ³⁷⁷⁻³⁹⁵peptide could specifically inhibit fibrin-mediated and not LPS-mediatedphosphorylation of Akt (FIG. 22). Overall, these results indicated thatinhibition of fibrin/Mac-1 interactions by the γ³⁷⁷⁻³⁹⁵ peptide inhibitsboth the morphological activation and the signaling cascade induced byfibrin-mediated activation of the Mac-1 microglia receptor.

Since genetic studies showed that the γ³⁷⁷⁻³⁹⁵ binding epitope offibrinogen to Mac-1 is not involved in coagulation (15), but issufficient to inhibit fibrin mediated microglia activation (FIGS.14-22), we examined whether blocking exclusively the inflammatoryproperties of fibrinogen in vivo using the γ³⁷⁷⁻³⁹⁵ would be sufficientto ameliorate Experimental Allergic Encephalitis (EAE). We thereforeexamined the effects of in vivo administration of the γ³⁷⁷⁻³⁹⁵ fibrinpeptide on microglia activation and clinical progression in an animalmodel for MS. We first assessed the effects of vaccination againstγ³⁷⁷⁻³⁹⁵ peptide. Vaccination with γ³⁷⁷⁻³⁹⁵ peptide before the inductionof EAE resulted in a significant reduction in disease penetrance andclinical symptoms. All control mice (15/15) developed clinical symptomsof EAE. By contrast, only 53% of the vaccinated mice (8/15) developedEAE. Moreover, mice vaccinated with the γ³⁷⁷⁻³⁹⁵ peptide showed anaverage clinical score of 1.1, while the control group showed a score of2.5 (FIG. 23, P<0.01). In a rotarod test, vaccinated mice showed a 76%increase in motor strength and coordination, when compared to thecontrol mice. Quantificative histopathological analysis showed reducedpathology in the brain (index of 1.5±2 vs. 2.3±1) and spinal cord (indexof 6.5±5.9 vs. 12±5.2) from γ³⁷⁷⁻³⁹⁵ peptide vaccinated mice as comparedto control mice.

Since vaccination is a preventive treatment, we further assessed whetherγ³⁷⁷⁻³⁹⁵ peptide would be beneficial if administered after the onset ofdisease. We administered intranasally (i.n.) 30 μg of γ³⁷⁷⁻³⁹⁵ peptidedaily after the first paralytic episode in remitting relapsing EAE (35).Intranasal delivery is a non-invasive delivery method, and results in ahigher degree of drug delivery to the nervous system and in a lowerdegree of systemic drug delivery to tissues such as liver and lymphnodes, when compared to intravenous (i.v.) delivery (38). Intranasaldelivery has been previously shown to be effective as a method of drugdelivery in EAE (39-41). γ³⁷⁷⁻³⁹⁵ peptide treated mice (n=14) incontrast to control mice (n=13) did not relapse (FIG. 24). Peptidetreated mice showed a 1.4-fold increase of motor functions when comparedto the control mice after the first relapse on day 29.

Immunohistochemical analysis using Mac-3, a microglia/macrophage marker,revealed reduction of activated cells in the peptide-treated (FIG. 26),as compared to control animals (FIG. 25). In addition, iNOS, a majorproduct of activated CD11b-positive microglia in EAE (42), was reducedin the γ³⁷⁷⁻³⁹⁵ peptide-treated animals (FIG. 26, inset). Quantificationof the histopathological analysis shows reduction in both Mac-3 andiNOS, while there are no major differences in T cell infiltrates (FIG.27). We further examined whether the γ³⁷⁷⁻³⁹⁵ peptide affected theperipheral immune response. FACS analysis on splenocytes of miceimmunized with PLP₁₃₉₋₁₅₁ using six markers for peripheral immune cells,namely CD4 and CD8 T cells, CD11b macrophages, CD11c dendritic cells,and CD19 and B220 B cells, did not reveal any differences betweenuntreated and γ³⁷⁷⁻³⁹⁵ peptide treated animals (FIG. 35), suggestingthat similar to systemic fibrin depletion (FIG. 34) the γ³⁷⁷⁻³⁹⁵ peptidedoes not affect the peripheral immune response. These results are inaccordance with prior studies where depletion of macrophages (43) ormicroglia (3) resulted in dramatic reduction of clinical symptoms in EAEeven in the presence of functional T cells.

Several studies have demonstrated both in vivo and in vitro thatfibrinogen interacts with different cellular receptors via nonoverlapping epitopes (for review see (16)). Fibrinogen regulates bloodcoagulation by engaging the platelet α_(IIb)β₃ integrin receptor via itsγ⁴⁰⁸⁻⁴¹¹ epitope, while it mediates inflammatory processes by engagingthe Mac-1 receptor via its γ³⁷⁷⁻³⁹⁵ epitope. As a result fibrinogenknock-in mice, where the γ³⁹⁰⁻³⁹⁶ Mac-1 binding site has been mutatedshow normal coagulation properties, such as platelet aggregation,thrombus formation and clotting time (15). To determine whether theγ³⁷⁷⁻³⁹⁵ peptide interfered with blood coagulation, we examined itseffects on the coagulative properties of fibrinogen both in vivo and invitro. As expected, the γ³⁷⁷⁻³⁹⁵ peptide does not affect coagulation inmice (FIG. 28) or prothrombin time (FIG. 29). Moreover, in an in vitrofibrin polymerization assay the γ³⁷⁷⁻³⁹⁵ peptide did not alter thepolymerization time of fibrin (FIG. 30). By GPRP peptide, an establishedinhibitor of clot formation (44), inhibits fibrin polymerization (FIG.30). Overall, these results show that in vivo delivery of the γ³⁷⁷⁻³⁹⁵peptide reduces the progression and severity of EAE by specificallytargeting microglia/macrophage activation in the CNS parenchyma withoutadverse hemorrhagic effects.

The present inventor has identified inhibition of fibrin/Mac-1interactions as a novel strategy for the attenuation of themicroglia/macrophage activation in the CNS. Histopathological studies inacute MS have identified perivenous lesions that appear to be driven byactivated microglia/macrophages sometimes in the absence of lymphocytes(52, 53). Although the activation of microglia and macrophages play acentral role in the pathogenesis of MS (3, 4, 43, 53), agents thatinhibit the microglia/macrophage response have not been developed (54).Although MS is a disease with profound lesion heterogeneity, BBBdisruption and microglia activation is a common feature for the fourdifferent subtypes of MS lesions (1). Therefore, targeting fibrin/Mac-1interactions can be beneficial in different MS subtypes as amicroglia-suppressive therapy and could be used in combinationaltherapies targeting other aspects of MS pathogenesis. In addition to MS,microglia activation is observed in a variety of neurodegenerativediseases characterized by BBB disruption, such as spinal cord injury,stroke and Alzheimer's disease (16). Therefore, targeting fibrin/Mac-1interactions represents a strategy for inhibiting microglia activationin neurodegenerative diseases. Since blocking fibrin/Mac-1 interactionsdoes not interfere with the physiological properties of fibrin in bloodcoagulation, this strategy can be applied in chronic diseases such asMS, without hemorrhagic side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosages for use in humans and othermammals. The dosage of such compounds lies preferably within a range ofcirculating plasma or other bodily fluid concentrations that include theED₅₀ with little or no toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized. For any compound of the invention, the therapeuticallyeffective dose can be estimated initially from cell culture assays. Adosage may be formulated in animal models to achieve a circulatingplasma concentration range that includes the IC₅₀ (the concentration ofthe test compound that achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful dosages in humans and other mammals.Compound levels in plasma may be measured, for example, by highperformance liquid chromatography.

The amount of a compound that may be combined with a pharmaceuticallyacceptable carrier to produce a single dosage form will vary dependingupon the host treated and the particular mode of administration. It willbe appreciated by those skilled in the art that the unit content of acompound contained in an individual dose of each dosage form need not initself constitute a therapeutically effective amount, as the necessarytherapeutically effective amount could be reached by administration of anumber of individual doses. The selection of dosage depends upon thedosage form utilized, the condition being treated, and the particularpurpose to be achieved according to the determination of those skilledin the art.

The dosage regime for treating a disease or condition with the compoundsof the invention is selected in accordance with a variety of factors,including the type, age, weight, sex, diet and medical condition of thepatient, the route of administration, pharmacological considerationssuch as activity, efficacy, pharmacokinetic and toxicology profiles ofthe particular compound employed, whether a compound delivery system isutilized and whether the compound is administered as a pro-drug or partof a drug combination. Thus, the dosage regime actually employed mayvary widely from subject to subject.

The compounds/polypeptides of the present invention may be formulated byknown methods for administration to a subject using several routes whichinclude, but are not limited to, parenteral, oral, topical, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, intercranial, and ophthalmic routes. The individual compoundsmay also be administered in combination with one or more additionalcompounds of the present invention and/or together with otherbiologically active or biologically inert agents. Such biologicallyactive or inert agents may be in fluid or mechanical communication withthe compound(s) or attached to the compound(s) by ionic, covalent, Vander Waals, hydrophobic, hydrophillic or other physical forces. It ispreferred that administration is localized in a subject, butadministration may also be systemic.

The compounds of the present invention may be formulated by anyconventional manner using one or more pharmaceutically acceptablecarriers and/or excipients. Thus, the compounds and theirpharmaceutically acceptable salts and solvates may be specificallyformulated for administration, e.g., by inhalation or insufflation(either through the mouth or the nose) or oral, buccal, parenteral orrectal administration. The compounds may take the form of charged,neutral and/or other pharmaceutically acceptable salt forms. Examples ofpharmaceutically acceptable carriers include, but are not limited to,those described in REMINGTON'S PHARMACEUTICAL SCIENCES (A. R. Gennaro,Ed.), 20th edition, Williams & Wilkins PA, USA (2000).

The compounds may also take the form of solutions, suspensions,emulsions, tablets, pills, capsules, powders, controlled- orsustained-release formulations and the like. Such formulations willcontain a therapeutically effective amount of the compound, preferablyin purified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the patient. Theformulation should suit the mode of administration.

The compound may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form in ampoules or inmulti-dose containers with an optional preservative added. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass, plastic or the like. Theformulation may take such forms as suspensions, solutions or emulsionsin oily or aqueous vehicles, and may contain formulatory agents such assuspending, stabilizing and/or dispersing agents.

For example, a parenteral preparation may be a sterile injectablesolution or suspension in a nontoxic parenterally acceptable diluent orsolvent (e.g., as a solution in 1,3-butanediol). Among the acceptablevehicles and solvents that may be employed are water, Ringer's solution,and isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed including synthetic mono- ordiglycerides. In addition, fatty acids such as oleic acid may be used inthe parenteral preparation.

Alternatively, the compound may be formulated in powder form forconstitution with a suitable vehicle, such as sterile pyrogen-freewater, before use. For example, a compound suitable for parenteraladministration may comprise a sterile isotonic saline solutioncontaining between 0.1 percent and 90 percent weight per volume of thecompound. By way of example, a solution may contain from about 0.1percent to about 20 percent, more preferably from about 0.55 percent toabout 17 percent, more preferably from about 0.8 to about 14 percent,and still more preferably about 10 percent of the compound. The solutionor powder preparation may also include a solubilizing agent and a localanesthetic such as lignocaine to ease pain at the site of the injection.Other methods of parenteral delivery of compounds will be known to theskilled artisan and are within the scope of the invention.

For oral administration, the compound may take the form of tablets orcapsules prepared by conventional means with pharmaceutically acceptableexcipients such as binding agents, fillers, lubricants anddisintegrants:

The tablets or capsules may optionally be coated by methods well knownin the art. If binders and/or fillers are used with the compounds of theinvention, they are typically formulated as about 50 to about 99 weightpercent of the compound. In one aspect, about 0.5 to about 15 weightpercent of disintegrant, and particularly about 1 to about 5 weightpercent of disintegrant, may be used in combination with the compound. Alubricant may optionally be added, typically in an amount of less thanabout 1 weight percent of the compound. Techniques and pharmaceuticallyacceptable additives for making solid oral dosage forms are described inMarshall, SOLID ORAL DOSAGE FORMS, Modern Pharmaceutics (Banker andRhodes, Eds.), 7:359-427 (1979). Other less typical formulations areknown in the art.

Liquid preparations for oral administration may take the form ofsolutions, syrups or suspensions. Alternatively, the liquid preparationsmay be presented as a dry product for constitution with water or othersuitable vehicle before use. Such liquid preparations may be prepared byconventional means with pharmaceutically acceptable additives such assuspending agents (e.g., sorbitol syrup, cellulose derivatives orhydrogenated edible fats); emulsifying agents (e.g., lecithin oracacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethylalcohol or fractionated vegetable oils); and/or preservatives (e.g.,methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparationsmay also contain buffer salts, flavoring, coloring, perfuming andsweetening agents as appropriate. Preparations for oral administrationmay also be formulated to achieve controlled release of the compound.Oral formulations preferably contain 10% to 95% compound. In addition,the compounds of the present invention may be formulated for buccaladministration in the form of tablets or lozenges formulated in aconventional manner. Other methods of oral delivery of compounds will beknown to the skilled artisan and are within the scope of the invention.

Controlled-release (or sustained-release) preparations may be formulatedto extend the activity of the compound and reduce dosage frequency.Controlled-release preparations can also be used to effect the time ofonset of action or other characteristics, such as blood levels of thecompound, and consequently affect the occurrence of side effects.

Controlled-release preparations may be designed to initially release anamount of a compound that produces the desired therapeutic effect, andgradually and continually release other amounts of the compound tomaintain the level of therapeutic effect over an extended period oftime. In order to maintain a near-constant level of a compound in thebody, the compound can be released from the dosage form at a rate thatwill replace the amount of compound being metabolized and/or excretedfrom the body. The controlled-release of a compound may be stimulated byvarious inducers, e.g., change in pH, change in temperature, enzymes,water, or other physiological conditions or molecules.

Controlled-release systems may include, for example, an infusion pumpwhich may be used to administer the compound in a manner similar to thatused for delivering insulin or chemotherapy to specific organs ortumors. Typically, using such a system, the compound is administered incombination with a biodegradable, biocompatible polymeric implant thatreleases the compound over a controlled period of time at a selectedsite. Examples of polymeric materials include polyanhydrides,polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinylacetate, and copolymers and combinations thereof. In addition, acontrolled release system can be placed in proximity of a therapeutictarget, thus requiring only a fraction of a systemic dosage.

The compounds of the invention may be administered by othercontrolled-release means or delivery devices that are well known tothose of ordinary skill in the art. These include, for example,hydropropylmethyl cellulose, other polymer matrices, gels, permeablemembranes, osmotic systems, multilayer coatings, microparticles,liposomes, microspheres, or the like, or a combination of any of theabove to provide the desired release profile in varying proportions.Other methods of controlled-release delivery of compounds will be knownto the skilled artisan and are within the scope of the invention.

The compound may also be formulated as a depot preparation. Suchlong-acting formulations may be administered by implantation (e.g.,subcutaneously or intramuscularly or intercranially) or by injection.Accordingly, the compounds may be formulated with suitable polymeric orhydrophobic materials such as an emulsion in an acceptable oil or ionexchange resins, or as sparingly soluble derivatives such as a sparinglysoluble salt. Other methods of depot delivery of compounds will be knownto the skilled artisan and are within the scope of the invention.

Various other delivery systems are known in the art and can be used toadminister the compounds of the invention. Moreover, these and otherdelivery systems may be combined and/or modified to optimize theadministration of the compounds of the present invention. Exemplaryformulations using the compounds of the present invention are describedbelow (the compounds of the present invention are indicated as theactive ingredient, but those of skill in the art will recognize thatpro-drugs and compound combinations are also meant to be encompassed bythis term):

In various embodiments, the present invention can also involve kits.Such kits can include the compounds/polypeptides/antibodies of thepresent invention and, in certain embodiments, instructions foradministration. When supplied as a kit, different components of acompound formulation can be packaged in separate containers and admixedimmediately before use. Such packaging of the components separately can,if desired, be presented in a pack or dispenser device which may containone or more unit dosage forms containing the compound/polyepeptide. Thepack may, for example, comprise metal or plastic foil such as a blisterpack. Such packaging of the components separately can also, in certaininstances, permit long-term storage without losing activity of thecomponents. In addition, if more than one route of administration isintended or more than one schedule for administration is intended, thedifferent components can be packaged separately and not mixed prior touse. In various embodiments, the different components can be packaged inone combination for administration together.

Kits may also include reagents in separate containers such as, forexample, sterile water or saline to be added to a lyophilized activecomponent packaged separately. For example, sealed glass ampules maycontain lyophilized polypeptide and in a separate ampule, sterile water,sterile saline or sterile each of which has been packaged under aneutral non-reacting gas, such as nitrogen. Ampules may consist of anysuitable material, such as glass, organic polymers, such aspolycarbonate, polystyrene, ceramic, metal or any other materialtypically employed to hold reagents. Other examples of suitablecontainers include bottles that may be fabricated from similarsubstances as ampules, and envelopes that may consist of foil-linedinteriors, such as aluminum or an alloy. Other containers include testtubes, vials, flasks, bottles, syringes, and the like. Containers mayhave a sterile access port, such as a bottle having a stopper that canbe pierced by a hypodermic injection needle. Other containers may havetwo compartments that are separated by a readily removable membrane thatupon removal permits the components to mix. Removable membranes may beglass, plastic, rubber, and the like.

In certain embodiments, kits can be supplied with instructionalmaterials. Instructions may be printed on paper or other substrate,and/or may be supplied as an electronic-readable medium, such as afloppy disc, mini-CD-ROM, CD-ROM, DVD-ROM, Zip disc, videotape, audiotape, and the like. Detailed instructions may not be physicallyassociated with the kit; instead, a user may be directed to an Internetweb site specified by the manufacturer or distributor of the kit, orsupplied as electronic mail.

The identified compounds treat, inhibit, control and/or prevent, or atleast partially arrest or partially prevent, nervous system disordersand can be administered to a subject at therapeutically effective dosesfor the inhibition, prevention, prophylaxis or therapy. The compounds ofthe present invention comprise a therapeutically effective dosage of apolypeptide, a term which includes therapeutically, inhibitory,preventive and prophylactically effective doses of the compounds of thepresent invention and is more particularly defined above. Without beingbound to any particular theory, applicants surmise that thesepharmaceutical compounds are effective in treatment when administered toa subject suffering from a nervous system disorder. The subject ispreferably an animal, including, but not limited to, mammals, reptilesand avians, more preferably horses, cows, dogs, cats, sheep, pigs, andchickens, and most preferably human.

“Antagonist” includes any molecule that partially or fully blocks,inhibits, or neutralizes a biological activity of endogenous Mac-1.Similarly, “agonist” includes any molecule that mimics a biologicalactivity of endogenous Mac-1. Molecules that can act as agonists orantagonists include Abs or Ab fragments, fragments or variants ofendogenous Mac-1, peptides, antisense oligonucleotides, small organicmolecules, etc.

To assay for antagonists, Mac-1 is added to, or expressed in, a cellalong with the compound to be screened for a particular activity. If thecompound inhibits the activity of interest in the presence of the Mac-1,that compound is an antagonist to the Mac-1; if Mac-1 activity isenhanced, the compound is an agonist.

Mac-1-expressing cells can be easily identified using any of thedisclosed methods. For example, antibodies that recognize the amino- orcarboxy-terminus of human Mac-1 can be used to screen candidate cells byimmunoprecipitation, Western blots, and immunohistochemical techniques,or flow cytometry.

Screening techniques well known to those skilled in the art can identifyMac-1 agonist or antagonist molecules. Examples of antagonists andagonists include: (1) small organic and inorganic compounds, (2) smallpeptides, (3) Abs and derivatives, (4) polypeptides closely related toMac-1, (5) antisense DNA and RNA, (6) ribozymes, (7) triple DNA helices,(8) siRNAs and (9) nucleic acid aptamers.

Small molecules that bind to the Mac-1 active site or other relevantpart of the polypeptide and inhibit the biological activity of the Mac-1are antagonists. Examples of small molecule antagonists include smallpeptides, peptide-like molecules, preferably soluble, and syntheticnon-peptidyl organic or inorganic compounds. These same molecules, ifthey enhance Mac-1 activity, are examples of agonists.

Almost any antibody that affects Mac-1's function can be a candidateantagonist or agonist. Examples of antibody antagonists includepolyclonal, monoclonal, single-chain, anti-idiotypic, chimeric Abs, orhumanized versions of such Abs or fragments. Abs may be from any speciesin which an immune response can be raised. Humanized Abs are alsocontemplated.

Alternatively, a potential antagonist or agonist may be a closelyrelated protein, for example, a mutated form of the Mac-1 thatrecognizes a Mac-1-interacting protein but imparts no effect, therebycompetitively inhibiting Mac-1 action. Alternatively, a mutated Mac-1may be constitutively activated and may act as an agonist.

“Percent (%) amino acid sequence identity” is defined as the percentageof amino acid residues that are identical with amino acid residues inthe disclosed polypeptide sequence in a candidate sequence when the twosequences are aligned. To determine % amino acid identity, sequences arealigned and if necessary, gaps are introduced to achieve the maximum %sequence identity; conservative substitutions are not considered as partof the sequence identity. Amino acid sequence alignment procedures todetermine percent identity are well known to those of skill in the art.Often publicly available computer software such as BLAST, BLAST2, ALIGN2or Megalign (DNASTAR) software is used to align peptide sequences. Thoseskilled in the art can determine appropriate parameters for measuringalignment, including any algorithms needed to achieve maximal alignmentover the full length of the sequences being compared.

When amino acid sequences are aligned, the % amino acid sequenceidentity of a given amino acid sequence A to, with, or against a givenamino acid sequence B (which can alternatively be phrased as a givenamino acid sequence A that has or comprises a certain % amino acidsequence identity to, with, or against a given amino acid sequence B)can be calculated as:

% amino acid sequence identity=X/Y·100

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program's or algorithm's alignment of A and Band Y is the total number of amino acid residues in B.

If the length of amino acid sequence A is not equal to the length ofamino acid sequence B, the % amino acid sequence identity of A to B willnot equal the % amino acid sequence identity of B to A.

Biologically active portions of fibrin/fibrinogen may have an amino acidsequence shown in SEQ ID NO: 1, or substantially homologous to SEQ IDNO: 1, and retains the functional activity of the protein of SEQ ID NO:1, yet differs in amino acid sequence due to natural allelic variationor mutagenesis. Other biologically active proteins comprise an aminoacid sequence at least about 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% homologous to the amino acid sequence of SEQID NO: 1 and retains the functional activity of SEQ ID NO: 1.

Such proteins which retain the functional activity of SEQ ID NO: 1 caninclude peptidomimetics. Thus, the invention also provides for reductionof a polypeptide having SEQ ID NO: 1 to generate mimetics, e.g. peptideor non-peptide agents, that are able to disrupt binding of Mac-1 toother proteins or molecules with which the native Mac-1 proteininteracts. Thus, the techniques described herein can also be used to mapwhich determinants of fibrin/fibrinogen protein participate in theintermolecular interactions involved in, e.g., binding offibrin/fibrinogen protein to other proteins which may function upstream(e.g., activators or repressors of fibrin/fibrinogen functionalactivity) of the fibrin/fibrinogen protein or to proteins or nucleicacids which may function downstream of the fibrin/fibrinogen protein,and whether such molecules are positively or negatively regulated by thefibrin/fibrinogen protein. To illustrate, the critical residues of anfibrin/fibrinogen protein which are involved in molecular recognitionof, e.g., fibrin/fibrinogen protein or other components upstream ordownstream of the fibrin/fibrinogen protein can be determined and usedto generate fibrin/fibrinogen protein-derived peptidomimetics whichcompetitively inhibit binding of the fibrin/fibrinogen protein to thatmoiety. By employing scanning mutagenesis to map the amino acid residuesof a fibrin/fibrinogen protein that are involved in binding otherextracellular proteins, peptidomimetic compounds can be generated whichmimic those residues of a native fibrin/fibrinogen protein. Suchmimetics may then be used to interfere with the normal function of anfibrin/fibrinogen protein.

For example, non-hydrolyzable peptide analogs of such residues can begenerated using benzodiazepine (see, e.g., Freidinger et al. inPeptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher.Leiden, Netherlands, 1988), azepine (e.g., see Huffman et al. inPeptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher:Leiden, Netherlands, 1988), substituted gamma lactam rings (Garvey etal. in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOMPublisher: Leiden, Netherlands, 1988), keto-methylene pseudopepitides(Ewenson et al. (1986) J. Med. Chem. 29:295; and Ewenson et al. inPeptides: Structure and Function (Proceedings of the 9th AmericanPeptide Symposium) Pierce Chemical Co. Rockland, Ill., 1985), beta-turndipeptide cores (Nagai et al. (1985) Tetrahedron Lett 26:647; and Satoet al. (1986) J. Chem. Soc. Perkin. Trans. 1: 1231), andbeta-aminoalcohols (Gordon et al. (1985) Biochem. Biophys. Res. Commun.126:419; and Dann et al. (1986) Biochem. Biophys. Res. Commun. 134:71).fibrin/fibrinogen proteins may also be chemically modified to createfibrin/fibrinogen protein derivatives by forming covalent or aggregateconjugates with other chemical moieties, such as glycosyl groups,lipids, phosphate, acetyl groups and the like. Covalent derivatives offibrin/fibrinogen protein can be prepared by linking the chemicalmoieties to functional groups on amino acid side chains of the proteinor at the N-terminus or at the C-terminus of the polypeptide.

Anti-Mac-1 Abs may further comprise humanized or human Abs. Humanizedforms of non-human Abs are chimeric Igs, Ig chains or fragments (such asF_(v), F_(ab), F_(ab′), F_((ab′)2) or other antigen-binding subsequencesof Abs) that contain minimal sequence derived from non-human 1 g.

Generally, a humanized antibody has one or more amino acid residuesintroduced from a non-human source. These non-human amino acid residuesare often referred to as “import” residues, which are typically takenfrom an “import” variable domain. Humanization is accomplished bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Such “humanized” Abs are chimeric Abs,wherein substantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Inpractice, humanized Abs are typically human Abs in which some CDRresidues and possibly some FR residues are substituted by residues fromanalogous sites in rodent Abs. Humanized Abs include human Igs(recipient antibody) in which residues from a complementary determiningregion (CDR) of the recipient are replaced by residues from a CDR of anon-human species (donor antibody) such as mouse, rat or rabbit, havingthe desired specificity, affinity and capacity. In some instances,corresponding non-human residues replace F_(v), framework residues ofthe human 1 g. Humanized Abs may comprise residues that are foundneither in the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody comprises substantiallyall of at least one, and typically two, variable domains, in which mostif not all of the CDR regions correspond to those of a non-human Ig andmost if not all of the FR regions are those of a human Ig consensussequence. The humanized antibody optimally also comprises at least aportion of an Ig constant region (F_(c)) typically that of a human 1 g.

Human Abs can also be produced using various techniques, including phagedisplay libraries and the preparation of human mAbs. Similarly,introducing human Ig genes into transgenic animals in which theendogenous Ig genes have been partially or completely inactivated can beexploited to synthesize human Abs. Upon challenge, human antibodyproduction is observed, which closely resembles that seen in humans inall respects, including gene rearrangement, assembly, and antibodyrepertoire.

Bi-specific Abs are monoclonal, preferably human or humanized, that havebinding specificities for at least two different antigens. For example,a binding specificity is Mac-1; the other is for any antigen of choice,preferably a cell-surface protein or receptor or receptor subunit.Traditionally, the recombinant production of bi-specific Abs is based onthe co-expression of two Ig heavy-chain/light-chain pairs, where the twoheavy chains have different specificities. Because of the randomassortment of Ig heavy and light chains, the resulting hybridomas(quadromas) produce a potential mixture of ten different antibodymolecules, of which only one has the desired bi-specific structure. Thedesired antibody can be purified using affinity chromatography or othertechniques.

To manufacture a bi-specific antibody, variable domains with the desiredantibody-antigen combining sites are fused to Ig constant domainsequences. The fusion is preferably with an Ig heavy-chain constantdomain, comprising at least part of the hinge, CH2, and CH3 regions.Preferably, the first heavy-chain constant region (CH1) containing thesite necessary for light-chain binding is in at least one of thefusions. DNAs encoding the Ig heavy-chain fusions and, if desired, theIg light chain, are inserted into separate expression vectors and areco-transfected into a suitable host organism.

The interface between a pair of antibody molecules can be engineered tomaximize the percentage of heterodimers that are recovered fromrecombinant cell culture. The preferred interface comprises at leastpart of the CH3 region of an antibody constant domain. In this method,one or more small amino acid side chains from the interface of the firstantibody molecule are replaced with larger side chains (e.g., tyrosineor tryptophan). Compensatory “cavities” of identical or similar size tothe large side chain(s) are created on the interface of the secondantibody molecule by replacing large amino acid side chains with smallerones (e.g., alanine or threonine). This mechanism increases the yield ofthe heterodimer over unwanted end products such as homodimers.

Bi-specific Abs can be prepared as full length Abs or antibody fragments(e.g., F_((ab′)2) bi-specific Abs). One technique to generatebi-specific Abs exploits chemical linkage. Intact Abs can beproteolytically cleaved to generate F_((ab′)2) fragments. Fragments arereduced with a dithiol complexing agent, such as sodium arsenite, tostabilize vicinal dithiols and prevent intermolecular disulfideformation. The generated F_(ab′) fragments are then converted tothionitrobenzoate (TNB) derivatives. One of the F_(ab′)-TNB derivativesis then reconverted to the F_(ab′)-thiol by reduction withmercaptoethylamine and is mixed with an equimolar amount of the otherF_(ab′)-TNB derivative to form the bi-specific antibody. The producedbi-specific Abs can be used as agents for the selective immobilizationof enzymes.

F_(ab′) fragments may be directly recovered from E. coli and chemicallycoupled to form bi-specific Abs. For example, fully humanizedbi-specific F_((ab′)2) Abs can be produced by methods known to those ofskill in the art. Each F_(ab′) fragment is separately secreted from E.coli and directly coupled chemically in vitro, forming the bi-specificantibody.

Various techniques for making and isolating bi-specific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, leucine zipper motifs can be exploited. Peptidesfrom the Fos and Jun proteins are linked to the F_(ab′) portions of twodifferent Abs by gene fusion. The antibody homodimers are reduced at thehinge region to form monomers and then re-oxidized to form antibodyheterodimers. This method can also produce antibody homodimers. The“diabody” technology provides an alternative method to generatebi-specific antibody fragments. The fragments comprise a heavy-chainvariable domain (V_(H)) connected to a light-chain variable domain(V_(L)) by a linker that is too short to allow pairing between the twodomains on the same chain. The V_(H) and V_(L) domains of one fragmentare forced to pair with the complementary V_(L) and V_(H) domains ofanother fragment, forming two antigen-binding sites. Another strategyfor making bi-specific antibody fragments is the use of single-chainF_(V) (sF_(V)) dimers. Abs with more than two valences are alsocontemplated, such as tri-specific Abs.

Abs of the invention, including polyclonal, monoclonal, humanized andfully human Abs, can be used therapeutically. Such agents will generallybe employed to treat or prevent a disease or pathology in a subject. Anantibody preparation, preferably one having high antigen specificity andaffinity generally mediates an effect by binding the target epitope(s).Generally, administration of such Abs may mediate one of two effects:(1) the antibody may prevent ligand binding, eliminating endogenousligand binding and subsequent signal transduction, or (2) the antibodyelicits a physiological result by binding an effector site on the targetmolecule, initiating signal transduction.

A therapeutically effective amount of an antibody relates generally tothe amount needed to achieve a therapeutic objective, epitope bindingaffinity, administration rate, and depletion rate of the antibody from asubject. Common ranges for therapeutically effective doses may be, as anonlimiting example, from about 0.1 mg/kg body weight to about 50 mg/kgbody weight. Dosing frequencies may range, for example, from twice dailyto once a week.

Anti-Mac-1 interacting molecules (such as aptamers) identified in otherassays, can be administered in pharmaceutical compositions as disclosed,infra, to treat various disorders. Abs that are internalized arepreferred when whole Abs are used as inhibitors. Liposomes may also beused as a delivery vehicle for intracellular introduction. Whereantibody fragments are used, the smallest inhibitory fragment thatspecifically binds to the epitope is preferred. For example, peptidemolecules can be designed that bind a preferred epitope based on thevariable-region sequences of a useful antibody. Such peptides can besynthesized chemically and/or produced by recombinant DNA technology.Formulations may also contain more than one active compound for aparticular treatment, preferably those with activities that do notadversely affect each other. The composition may comprise an agent thatenhances function, such as a cytotoxic agent, cytokine, chemotherapeuticagent, or growth-inhibitory agent.

The active ingredients can also be entrapped in microcapsules preparedby coacervation techniques or by interfacial polymerization; forexample, hydroxymethylcellulo se or gelatin-microcapsules andpoly-(methylmethacrylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nanoparticles, and nanocapsules) or in macroemulsions.The formulations to be used for in vivo administration are highlypreferred to be sterile. This is readily accomplished by filtrationthrough sterile filtration membranes or any of a number of techniques.

Sustained-release preparations may also be prepared, such assemi-permeable matrices of solid hydrophobic polymers containing theantibody, which matrices are in the form of shaped articles, e.g.,films, or microcapsules. Examples of sustained-release matrices includepolyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate),or poly(vinylalcohol)), polylactides, copolymers of L-glutamic acid andγ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as injectable microspherescomposed of lactic acid-glycolic acid copolymer, andpoly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days, certain hydrogels release proteins for shorter timeperiods and may be preferred.

EXAMPLES

Aspects of the present teachings may be further understood in light ofthe following examples, which should not be construed as limiting thescope of the present teachings in any way. Materials and methods used inthe examples include the following:

Animals. Female SJL/J and C57BL/6 mice (6 weeks old) were purchased fromthe Jackson Laboratory (Bar Harbor, Me.) or Harlan Sprague Dawley(Indianapolis, Ind.). Female Fibγ^(390-396A) mice (6 weeks old) weregenerated from double heterozygous matings to produce homozygousFibγ^(390-396A) mice (15) and Fibγ^(WT) littermate mice as controls.Fibγ^(390-396A) mice were backcrossed six generations to C57BI/6 mice(15).

Induction of EAE. EAE was induced in 6 week old female SJL/J or C57BL/6mice by subcutaneous immunization with 150 μg PLP₁₃₉₋₁₅₁ (HSLGKWLGHPDKF)(SEQ ID NO: 5). American Peptide Company and Azco Pharmchem, San Jose,Calif.) or 50 μg MOG₃₅₋₅₅ (MEVGWYRSPFSRVVHLYRDGK) (SEQ ID NO: 6),Sigma-Aldrich, St. Louis, Mo.) in complete Freund's adjuvant(Sigma-Aldrich, St. Louis, Mo.) supplemented with 200 ng heatinactivated mycobacterium tuberculosis H37Ra (Difco Laboratories,Detroit, Mich.). Mice were injected intravenously with 200 ng pertussistoxin (Sigma-Aldrich, St. Louis, Mo.) on days 0 and 2 of theimmunization. Mice were scored daily. 0: no symptoms, 1: loss of tailtone, 2: ataxia, 3: hindlimb paralysis, 4: hindlimb and forelimbparalysis, 5: moribund Data are represented as the mean clinical scoreand are mean±SEM. Statistical calculations were made by using Student'st test.

Systemic defibrinogenation. Mice were depleted of fibrinogen asdescribed (55). The pumps deliver 0.5 μl/hour, thus the mice received2.4 U ancrod/day. In control animals, buffer-filled mini-pumps wereimplanted.

Fibrinogen γ³⁷⁷⁻³⁹⁵ peptide vaccination. Vaccination was performed asdescribed (56) with the following modifications. Five week old femaleSJL/J mice were immunized with 200 μg fibrinogen γ³⁷⁷⁻³⁹⁵ peptide(YSMKETTMKIIPFNRLSIG) (SEQ ID NO: 2), Azco Pharmchem, San Jose, Calif.)emulsified with an equal volume of incomplete freund's adjuvant(Sigma-Aldrich, St. Louis, Mo.). Mice were immunized four times inalternating rear flanks over the course of two weeks. Control animalswere immunized with incomplete Freund's adjuvant.

Intranasal γ³⁷⁷⁻³⁹⁵ peptide administration. Fibrinogen γ³⁷⁷⁻³⁹⁵ peptide(YSMKETTMKIIPFNRLSIG) (SEQ ID NO: 2), Azco Pharmchem, San Jose, Calif.)was resuspended in 0.9% NaCl at a concentration of 3 mg/mL. Peptideswere aliquoted as single doses and stored at 20° C. Mice wereadministered 5 μL of peptide or 0.9% NaCl, as a control, in each naredaily using a P10 micropipettor (Gilson, Middleton, Wis.) beginningafter the peak of the first paralytic episode.

Histopathology. Histopathologic analysis and quantification ofinflammation and demyelination in mouse tissue was performed on cryostator paraffin sections as described (57). Sections were stained with luxolfast blue and nuclear red. Spinal cord sections were fixed with 2% PFAfor 10 minutes at 4° C. and immunostained with a sheep anti-fibrinogenantibody (1:200, US Biological, Swampscott, Mass.), rat anti-CD11b (1:5,Chemicon), von Willebrand Factor (1:1000, Dakocytomation, Glostrup,Denmark), iNOS (polyclonal Chemicon, Temecula, Calif., 1:750), Mac-3(rat-anti-mouse, Pharmingen, San Diego, Calif., 1:200), CNPase(monoclonal, Sternberger monoclonals, Lutherville, Md., 1:2000). Forhuman MS, paraffin embedded material was obtained from the Archives ofthe Center for Brain Research, Medical University of Vienna. Doubleimmunofluorescence was performed with antibodies against CD68 andfibrin. Images were collected using an Axioplan 2 Zeiss microscope withan Axiocam HRc camera or were processed for confocal microscopy usingOlympus and Zeiss confocal microscopes.

Rotarod behavior test. The rotarod assay was performed on a TSEacceleratingrRotarod apparatus (TSE Technical and Scientific EquipmentGmbH) as described (58). Rotarod assays were conducted with a 300 secondmaximum time limit and means were collected for at least three trials.Statistical calculations were made by using Students t test.

Culture of primary microglia cells. Microglia were isolated fromcultures of mixed cortical cells as described (59). In brief, corticesfrom P1 mice were isolated and digested with trypsin (0.4%) for 20minutes at 37° C. Cells from 4 pups were plated onto PDL-coated 75 cm2tissue-culture flasks. The culture medium (DMEM, 10% heat-inactivatedFBS, and 1% Pen/Strep) was changed on day 2. After 2-3 weeks in culture,the microglia were removed by the addition of 12 mM lidocaine(Sigma-Aldrich, St. Louis, Mo.) and orbital shaking (180 rpm) for 20minutes at 37° C. The cells were centrifuged (350×G) and the pellet wasresuspended in complete medium and plated onto PDL-coated tissue culturedishes. Microglia cell cultures were >95% pure as determined with threedifferent markers, IsoB4, IBA-1 and CD11b. To immobilize fibrinogen,tissue culture dishes were incubated overnight with 50 μg/mL fibrinogen(Calbiochem, San Diego, Calif.) in 20 mM Tris (pH 7.5) and 0.1 M NaCl at37° C. for all fibrinogen treatments. To serve as positive controls, LPS(Sigma-Aldrich, St. Louis, Mo.) was added to the media at 1 μg/mL.

Phagocytosis assay. Phagocytosis assays were performed on both primarymurine microglia and a murine microglia cell line (BV2). BV2 cells arean established cell line used to study microglia responses (46). BV2cells were routinely passaged in DMEM, 20% heat inactivated FBS, and 1%Pen/Strep. Microglia phagocytosis was assessed using the Vybrantphagocytosis assay kit (Molecular Probes, Eugene, Oreg.) according tothe manufacturer's recommendations. Microglia were cultured in 96-wellplates at 5,000 cells/well for 36 hours at 37° C. Blocking experimentsincluded the addition of LY294002 (1 μM, Cell Signaling, Beverly,Mass.), rat anti-CD 11b (10 μg/mL, eBioscience, San Diego, Calif.), ratanti-TLR4 (10 μg/mL, eBioscience, San Diego, Calif.) or rat IgG (10μg/mL, Jackson ImmunoResearch Laboratories, West Grove, Pa.) to themedia. Results were obtained from four separate experiments performed intriplicates. Statistical calculations were made by using Student'st-test.

Morphometry. Primary microglia were used for all morphologic analysis.Cells were plated on coated fibrinogen or in the presence of LPS for 72hours. Microglia were fixed with methanol and immunostained withIsolectin B4 (1:300, Sigma-Aldrich, St. Louis, Mo.). Activated microgliawere quantificated as those cells larger than 2,000 μm². Blockingexperiments involved the daily administration of rat anti-CD11b (10μg/mL, eBioscience, San Diego, Calif.), γ³⁷⁷⁻³⁹⁵ peptide (200 μM) or ratIgG (10 μg/mL, Jackson ImmunoResearch Laboratories, West Grove, Pa.).Results were obtained from six separate experiments performed induplicates. 250 cells per condition were counted for each individualexperiment. Statistical calculations were made by using Student's ttest.

Endotoxin detection assay. Fibrinogen samples were tested forcontaminating endotoxins by a Limulus Amebocyte Lysate assay (E-TOXATEkit; Sigma-Aldrich, St. Louis, Mo.). Fibrinogen-treated samples hadundetectable endotoxin levels (<0.5 endotoxin units).

Immunoblots. Western blots were performed using standard protocols (55).Microglia were serum-starved overnight and plated on fibrinogen or withLPS for 6 hours. Lysates were electrophoresed on 4-12% gradient SDS-PAGEgels and probed with phospho and total Akt antibodies (1:1000, CellSignaling, Beverly, Mass.). RhoA activation was performed as described(60). Briefly, 2×107 microglia were plated on fibrinogen for 6 hours orwith LPS for 10 minutes. Cell lysates were incubated with GST-Rhotekinagarose beads (kindly provided by Dr. Joan Heller Brown, UCSD) for 45minutes at 4° C. The beads were washed, resuspended in Laemmli samplebuffer, and electophoresed on a 15% SDS-PAGE gel. Activated and totalRho were detected with mouse anti-RhoA (1:500, Santa Cruz Biotechnology,Santa Cruz, Calif.). Densitometry was performed using the NIH ScionImage software using three blots from three separate experiments.

Deconvolution microscopy. Fluorescent images were obtained as described(60). Primary microglia were stimulated with fibrinogen as describedabove. Cells were immunostained with antibodies to total actin (1:100,Sigma-Aldrich, St. Louis, Mo.) or β-tubulin (1:100, Sigma-Aldrich, St.Louis, Mo.).

Isolation of mouse splenocytes and T cell proliferation assay. Spleenswere removed from control and fibrin-depleted mice after induction ofPLP₁₃₉₋₁₅₁ EAE and washed in PBS. Spleens were mechanically dissociatedwith sterile blades and filtered through 70 μm nylon screens.Splenocytes were pelleted and washed with an erythrocyte lysing solution(Biolegend, San Diego, Calif.). Cells were washed twice with PBS,counted and seeded at a density of 0.5×10⁶ cells/96-well. Cells fromboth control and fibrin-depleted mice were either untreated orstimulated with 20 μg/mL PLP₁₃₉₋₁₅₁ and proliferation was assayed byBrdU incorporation (Roche Applied Sciences, Indianapolis, Ind.)according to the manufacturer's instructions.

FACS Analysis. Primary mouse splenocytes were isolated from control andγ³⁷⁷⁻³⁹⁵ peptide-treated mice, immunized with PLP139-151, on day 29after immunization. Cells were immunostained with antibodies to CD4,CD8, CD11b, CD11c, CD19, B220 (1:100, Biolegend, San Diego, Calif.) andanalyzed on a Becton Dickinson FACSCalibur flow cytometer.

Hematological Analyses. Citrated plasma was prepared from miceadministered intranasally 30 μg or 90 μg of γ³⁷⁷⁻395 peptide or salinecontrol daily for seven days. Plasma clotting times were measured bycombining 10 μl of citrated plasma with 10 μl of 2 U/ml bovine thrombin(Enzyme Research Laboratories, South Bend, Ind., USA) and 40 mM CaCl₂ ina weigh boatfloating in a 37° C. water bath. Time to clot was determinedby mixing with a toothpick. Plasma thrombin times were established asdescribed previously (15). Fibrin polymerization was evaluated bystandard turbidity assays using plasma. Briefly, citrate plasma (dilutedtenfold in 20 mM HEPES, pH 7.4, containing 0.15 M NaCl and 5 mM-aminocaproic acid) was combined with bovine thrombin (final concentration 0.2U/ml; Enzyme Research Laboratories, South Bend, Ind., USA), and Ca²⁺ (10mM), and OD₃₅₀ measurements were taken every 30 seconds.

Example 1

This example illustrates that fibrinogen directly activates microglia,resulting in cytoskeletal rearrangement and increased phagocytosis.Fibrinogen directly activates microglia resulting in cytoskeletalrearrangements and increased phagocytosis. In this example, we firsttested the effects of fibrinogen on pure primary microglia cells.Immobilized fibrinogen had a dramatic effect on microglia activationcharacterized by an increase in cell size (FIG. 1, right column), whencompared to untreated controls (FIG. 1, left column). Quantificationrevealed a 85.5±1.4% of activated microglia after fibrinogen treatmentvs. 8.2±3.4% in untreated cells (FIGS. 5-10; P<0.001). By contrast toimmobilized fibrinogen, soluble fibrinogen did not induce changes inmicroglia morphology (data not shown). Lipopolysaccharide (LPS) was usedas a positive control (FIGS. 1-10). Using an endotoxin determinationassay, we verified that there was no LPS contamination in thefibrinogen-treated cultures. To assess the functional effect of thismorphologic activation, we performed phagocytosis assays. Fibrinogenstimulation resulted in a 65.3% increase in phagocytosis as compared tountreated controls (P<0.05), while LPS incubation, by comparison,increased phagocytosis by 39.1% (P<0.05) (FIGS. 11-13). Previous studieshave demonstrated that dynamic rearrangements of both the actin (26) andmicrotubule (27) networks are critical for phagocytosis. Deconvolutionmicroscopy showed that fibrinogen stimulation resulted in dramaticrearrangement of the microglial cytoskeleton as determined byimmunostaining of actin and (β-tubulin (FIGS. 14-19). Overall, theseresults indicate that fibrinogen induces microglia differentiation intoa phagocytic state.

Example 2

This example illustrates that fibrinogen can activate microglia via theMac-1 integrin receptor Mac-1 orchestrates the innate immune response,by regulating phagocyte adhesion, migration, and engulfment ofcomplement-opsonized particles (28). M1/70, an antibody which blocksbinding of fibrinogen to CD11b (24, 29), inhibited fibrin-mediatedmicroglia activation (FIGS. 20-22). Quantification showed 71±4.9% ofactivated microglia in fibrinogen treated cells vs. 18±0.7% in cellstreated with fibrinogen in the presence of M1/70 (FIGS. 23-27, P<0.01).The inhibitory effect of M1/70 was specific for fibrinogen and did notaffect LPS-mediated activation of microglia (FIG. 6). Moreover, blockingCD11b reduced phagocytosis of fibrinogen-treated cells by 40% (P<0.05)(FIGS. 28-30). Previous studies have established that the Toll-likeReceptor-4 (TLR4) LPS receptor mediates LPS-induced microglia activation(30, 31). By contrast, blocking TLR-4 did not affect fibrinogen-mediatedphagocytosis, suggesting that Mac-1 was the major receptor to inducefibrinogen-mediated phagocytosis (FIGS. 28-30).

Example 3

This example illustrates fibrinogen induces increases in RhoA and Aktphosphorylation in microglia. RhoA and PI3K are the two major signalingpathways downstream of Mac-1 that mediate the cytoskeletalrearrangements for the induction of phagocytosis (28, 32, 33).Fibrinogen applied as described above induced a 1.9-fold increase ofactive RhoA and a 24-fold increase in Akt phosphorylation in microglia(FIG. 31). Furthermore, blocking the PI3K signaling pathway usingLY294002, inhibited the fibrin-induced increase of phagocytosis inmicroglia (FIGS. 28-30, P<0.01), suggesting that PI3K is downstream offibrin/Mac-1 signaling in microglia cells. This is the first evidencesuggesting that fibrinogen can induce Mac-1-dependent signaling inmicroglia.

Example 4

This example illustrates induction of phagocytosis in microglia.Analysis of demyelinated spinal cords after induction of PLP₁₃₉₋₁₅₁experimental autoimmune encephalomyelitis (EAE) in mice showed thatCD11b-positive microglia (FIG. 9, top right panel), were surrounded byfibrin (FIG. 9, top left panel). Similarly, analysis of acutedemyelinating lesions of human MS showed that fibrin (FIG. 10, middlepanel) surrounded activated microglia cells (FIG. 10, top panel).Overall these results show that fibrinogen/CD11b signaling inducesphagocytosis in microglia and demonstrate both in EAE and in human MSthe presence of this ligand/receptor system on active microglia withininflammatory demyelinating lesions.

Example 5

This example illustrates that fibrin depletion inhibits microgliaactivation in vivo and attenuates inflammatory demyelination. In thisexample, we further examined whether fibrin is required for theactivation of Mac-1 positive cells in vivo. In these experiments, weadministered the anticoagulant ancrod (34) in an establishedremitting-relapsing model of PLP₁₃₉₋₁₅₁ EAE (35) after the developmentof the first paralytic incidence. At the time of the first relapse,untreated mice show dramatic activation of CD11b-positive cellscharacterized by thick processes (FIG. 11, left column). By contrast, inancrod-treated mice CD11b-positive cells appear with ramified morphology(FIG. 11, right column) resembling CD11b-positive cells in the normalspinal cord (FIG. 32). Single channel images are shown in FIG. 32.Untreated mice showed extensive inflammatory demyelinating lesions inthe cerebellum (FIG. 33, asterisk) and spinal cord (FIG. 33, arrows). Bycontrast, in ancrod-treated mice inflammatory demyelination wasdramatically decreased (FIG. 33). Ancrod treatment resulted in decreaseddemyelination by 7-fold in the cerebellum and by 2-fold in the spinalcord. Splenocytes isolated from untreated and ancrod-treated mice showedno difference in proliferation upon stimulation with PLP_(139·151) (FIG.S4), further suggesting that CD11b-positive cells are the major celltarget of fibrin in the nervous system. Fibrin-depleted mice recoveredfaster from the first paralytic incidence and, in contrast to controlmice, never relapsed (FIG. 12). In a rotarod behavioral test, performedbefore the first relapse of the untreated group, fibrin-depleted miceshowed a 3-fold increase of motor strength and coordination whencompared to the untreated group (FIG. 13). Overall, these resultssuggest that fibrin is a major contributor to the local activation ofCD11b-positive cells in vivo. In addition, this is the firstdemonstration that an anti-coagulant may improve clinical symptoms andreduce inflammatory demyelination when administered after the onset ofparalytic symptoms.

Example 6

This example illustrates that Fibγ^(390-396A) mice have reduced clinicalscores and inflammation. To examine the contribution of fibrinogen/Mac-1signaling in inflammatory demyelination in vivo, we utilized mice with aknock-in mutation of seven amino acids at residues (N₃₉₀RLSIGE₃₉₆) (SEQID NO: 3) to alanine residues (A₃₉₀AAAAAA₃₉₆) (SEQ ID NO: 4) thatabolishes fibrinogen binding to the Mac-1 receptor (15). These mice,termed Fibγ^(390-396A), and their age- and sex-matched littermatecontrols (Fibγ^(wT)) were in C57BI/6 background and were immunized withMOG₃₅₋₅₅ peptide (FIG. 14). Fib γ^(390-96A) mice showed an averageclinical score of 2.2 (ataxia) at day 17 after immunization, while theFibγ^(WT) mice developed clinical symptoms of paralysis, showing anaverage score of 3.7 (hind limb paralysis) (P<0.01). In the Fibγ^(WT)mouse group 73% of Fibγ^(WT) mice (11/15) were paralyzed, by contrast to24% of Fibγ^(390-396A) mice (4/17) (clinical score>3) (FIG. 15), furtherdemonstrating decreased severity of EAE. Fibγ^(390-396A) mice showed a1.5-fold increase in motor skills, when compared to Fibγ^(WT) mice(Fibγ^(390-396A), 269±9 sec vs. Fibγ^(WT), 169±23 sec, P<0.05) (FIG.18). Lesions were decreased 3-fold in Fibγ^(390-396A) mice, whencompared to Fibγ^(WT) (FIG. 16). In accordance, spinal cord sectionsshowed a decrease of IsoB4-positive cells in Fibγ^(390-396A) mice, whencompared to Fibγ^(WT) controls (FIG. 19). Decrease in clinical severitywas also observed at later time points after the immunization (FIGS. 14and 15). In addition, 27% Fibγ^(WT) mice (4/15) died by contrast to only6% of the Fibγ^(390-396A) mice (1/17) (FIG. 17). Overall, these resultssuggest that the γ³⁹⁰⁻³⁹⁶ binding site of fibrinogen to Mac-1 regulatesthe severity of inflammatory demyelination.

Example 7

This example illustrates that the fibrin γ³⁷⁷⁻³⁹⁵ peptide blocksmicroglia activation in vitro. Both the in vitro (FIGS. 2-10) and invivo experiments (FIGS. 14-19) show that fibrinogen signaling throughthe Mac-1 receptor contributes to microglia activation and regulates theseverity of inflammatory demyelination in EAE. Biochemical studiesidentified that the γ₃₇₇ YSMKKTTMKIIPFNRLTIG₃₉₅ (SEQ ID NO: 1) peptideblocks fibrin binding to Mac-1 (29). The γ³⁷⁷⁻³⁹⁵ is a cryptic epitopein the fibrinogen molecule and is exposed only when fibrinogen isimmobilized or converted to fibrin (25, 36). We used 200 μM of peptide,a concentration shown to inhibit adhesion of Mac-1 overexpressing cellsto immobilized fibrinogen (37), to examine whether the γ³⁷⁷⁻³⁹⁵ peptidecould inhibit microglia activation. Fibrinogen treatment resulted in71±4.9% of activated microglia, when compared to 38.3±9.8% afteraddition of the γ³⁷⁷⁻³⁹⁵ peptide (P<0.05) (FIGS. 20-21), suggesting thatthe γ³⁷⁷⁻³⁹⁵ peptide reduced fibrin-mediated microglia activation. Theγ³⁷⁷⁻³⁹⁵ peptide did not affect the activation state of untreatedmicroglia and did not inhibit LPS-mediated microglia activation (FIG.21), further suggesting the specificity of γ³⁷⁷⁻³⁹⁵ to block activationof Mac-1 by fibrin. In accordance, examination of Akt phosphorylationshowed that the γ³⁷⁷⁻³⁹⁵ peptide could specifically inhibitfibrin-mediated and not LPS-mediated phosphorylation of Akt (FIG. 22).Overall, these results suggest that inhibition of fibrin/Mac-1interactions by the γ³⁷⁷⁻³⁹⁵ peptide inhibits both the morphologicalactivation and the signaling cascade induced by fibrin-mediatedactivation of the Mac-1 microglia receptor.

Example 8

This example illustrates that prophylactic or therapeutic administrationof the fibrin γ³⁷⁷⁻³⁹⁵ peptide suppresses EAE and inhibits microgliaactivation in vivo. Since genetic studies showed that the γ³⁷⁷⁻³⁹⁵binding epitope of fibrinogen to Mac-1 is not involved in coagulation(15), but is sufficient to inhibit fibrin-mediated microglia activation(FIGS. 14-22), we examined whether blocking exclusively the inflammatoryproperties of fibrinogen in vivo using the γ³⁷⁷⁻³⁹⁵ would be sufficientto ameliorate EAE. We therefore examined the effects of in vivoadministration of the γ³⁷⁷⁻³⁹⁵ fibrin peptide on microglia activationand clinical progression in an animal model for MS. We first assessedthe effects of vaccination against γ³⁷⁷⁻³⁹⁵ peptide. Vaccination withγ³⁷⁷⁻³⁹⁵ peptide before the induction of EAE resulted in a significantreduction in disease penetrance and clinical symptoms. All control mice(15/15) developed clinical symptoms of EAE. By contrast, only 53% of thevaccinated mice (8/15) developed EAE. Moreover, mice vaccinated with theγ³⁷⁷⁻³⁹⁵ peptide showed an average clinical score of 1.1, while thecontrol group showed a score of 2.5 (FIG. 23, P<0.01). In a rotarodtest, vaccinated mice showed a 76% increase in motor strength andcoordination, when compared to the control mice. Quantitativehistopathological analysis showed reduced pathology in the brain (indexof 1.5±2 vs. 2.3±1) and spinal cord (index of 6.5±5.9 vs. 12±5.2) fromγ³⁷⁷⁻³⁹⁵ peptide vaccinated mice as compared to control mice.

Since vaccination is a preventive treatment, we further assessed whetherγ³⁷⁷⁻³⁹⁵ peptide would be beneficial if administered after the onset ofdisease. We administered intranasally (i.n.) 30 μg of γ³⁷⁷⁻³⁹⁵ peptidedaily after the first paralytic episode in remitting relapsing EAE (35).Intranasal delivery is a non-invasive delivery method, and results in ahigher degree of drug delivery to the nervous system and in a lowerdegree of systemic drug delivery to tissues such as liver and lymphnodes, when compared to intravenous (i.v.) delivery (38). Intranasaldelivery has been previously shown to be effective as a method of drugdelivery in EAE (39-41). γ³⁷⁷⁻³⁹⁵ peptide treated mice (n=14) incontrast to control mice (n=13) did not relapse (FIG. 24). Peptidetreated mice showed a 1.4-fold increase of motor functions when comparedto the control mice after the first relapse on day 29.

Immunohistochemical analysis using Mac-3, a microglia/macrophage marker,revealed reduction of activated cells in the peptide-treated (FIG. 26),as compared to control animals (FIG. 25). In addition, iNOS, a majorproduct of activated CD11b-positive microglia in EAE (42), was reducedin the γ³⁷⁷⁻³⁹⁵ peptide-treated animals (FIG. 26, inset). Quantificationof the histopathological analysis shows reduction in both Mac-3 andiNOS, while there are no major differences in T cell infiltrates (FIG.27). We further examined whether the γ³⁷⁷⁻³⁹⁵ peptide affected theperipheral immune response. FACS analysis on splenocytes of miceimmunized with PLP₁₃₉₋₁₅₁ using six markers for peripheral immune cells,namely CD4 and CD8 T cells, CD11b macrophages, CD11c dendritic cells,and CD19 and B220 B cells, did not reveal any differences betweenuntreated and γ³⁷⁷⁻³⁹⁵ peptide treated animals (FIG. 35), suggestingthat similar to systemic fibrin depletion (FIG. 34) the γ³⁷⁷⁻³⁹⁵ peptidedoes not affect the peripheral immune response. These results are inaccordance with prior studies where depletion of macrophages (43) ormicroglia (3) resulted in dramatic reduction of clinical symptoms in EAEeven in the presence of functional T cells.

Example 9

This example illustrates that fibrin γ³⁷⁷⁻³⁹⁵ peptide does not affectthe coagulation properties of fibrinogen. Several studies havedemonstrated both in vivo and in vitro that fibrinogen interacts withdifferent cellular receptors via non overlapping epitopes (for reviewsee (16)). Fibrinogen regulates blood coagulation by engaging theplatelet α_(11b)β₃ integrin receptor via its γ⁴⁰⁸⁻⁴¹¹ epitope, while itmediates inflammatory processes by engaging the Mac-1 receptor via itsγ³⁷⁷⁻³⁹⁵ epitope. As a result fibrinogen knock-in mice, where theγ³⁹⁰⁻³⁹⁶ Mac-1 binding site has been mutated show normal coagulationproperties, such as platelet aggregation, thrombus formation andclotting time (15). To determine whether the γ³⁷⁷⁻³⁹⁵ peptide interferedwith blood coagulation, we examined its effects on the coagulativeproperties of fibrinogen both in vivo and in vitro. As expected, theγ³⁷⁷⁻³⁹⁵ peptide does not affect coagulation in mice (FIG. 28) orprothrombin time (FIG. 29). Moreover, in an in vitro fibrinpolymerization assay the γ³⁷⁷⁻³⁹⁵ peptide did not alter thepolymerization time of fibrin (FIG. 30). By contrast, the GPRP peptide,an established inhibitor of clot formation (44), inhibits fibrinpolymerization (FIG. 30). Overall, these results show that in vivodelivery of the γ³⁷⁷⁻³⁹⁵ peptide reduces the progression and severity ofEAE by specifically targeting microglia/macrophage activation in the CNSparenchyma without adverse hemorrhagic effects.

Other Embodiments

The detailed description set-forth above is provided to aid thoseskilled in the art in practicing the present invention. However, theinvention described and claimed herein is not to be limited in scope bythe specific embodiments herein disclosed because these embodiments areintended as illustration of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description which do not depart from thespirit or scope of the present inventive discovery. Such modificationsare also intended to fall within the scope of the appended claims.

REFERENCES CITED

All publications, patents, patent applications and other referencescited in this application are incorporated herein by reference in theirentirety for all purposes to the same extent as if each individualpublication, patent, patent application or other reference wasspecifically and individually indicated to be incorporated by referencein its entirety for all purposes. Citation of a reference herein shallnot be construed as an admission that such is prior art to the presentinvention. Publications incorporated herein by reference in theirentirety include:

-   1. Lassmann, H., W. Bruck, and C. Lucchinetti 2001. Heterogeneity of    multiple sclerosis pathogenesis: implications for diagnosis and    therapy. Trends Mol Med 7:115-121.-   2. Platten, M., and L. Steinman. 2005. Multiple sclerosis: trapped    in deadly glue. Nat Med 11:252-253.-   3. Heppner, F. L., M. Greter, D. Marino, J. Falsig, G. Raivich, N.    Hovelmeyer, A. Waisman, T. Rulicke, M. Prinz, J. Priller, B. Becher,    and A. Aguzzi. 2005. Experimental autoimmune encephalomyelitis    repressed by microglial paralysis. Nat Med 11:146-152.-   4. Jack, C., F. Ruffini, A. Bar-Or, and J. P. Antel. 2005. Microglia    and multiple sclerosis. J Neurosci Res 81:363-373.-   5. Minagar, A., and J. S. Alexander. 2003. Blood-brain barrier    disruption in multiple sclerosis. Mult Scler 9:540-549.-   6. Kermode, A. G., A. J. Thompson, P. Tofts, D. G. MacManus, B. E.    Kendall, D. P. Kingsley, I. F. Moseley, P. Rudge, and W. I.    McDonald. 1990. Breakdown of the blood-brain barrier precedes    symptoms and other MRI signs of new lesions in multiple sclerosis.    Pathogenetic and clinical implications. Brain 113:1477-1489.-   7. Nimmerjahn, A., F. Kirchhoff, and F. Helmchen. 2005. Resting    Microglial Cells Are Highly Dynamic Surveillants of Brain Parenchyma    in Vivo. Science 308:1314-1318.-   8. Claudio, L., C. S. Raine, and C. F. Brosnan. 1995. Evidence of    persistent blood-brain barrier abnormalities in chronic-progressive    multiple sclerosis. Acta Neuropathol 90:228-238.-   9. Kwon, E. E., and J. W. Prineas. 1994. Blood-brain barrier    abnormalities in longstanding multiple sclerosis lesions. An    immunohistochemical study. J Neuropathol Exp Neurol 53:625-636.-   10. Wakefield, A. J., L. J. More, J. Difford, and J. E.    McLaughlin. 1994. Immunohistochemical study of vascular injury in    acute multiple sclerosis. J Clin Pathol 47:129-133.-   11. Gay, F. W., T. J. Drye, G. W. Dick, and M. M. Esiri. 1997. The    application of multifactorial cluster analysis in the staging of    plaques in early multiple sclerosis. Identification and    characterization of the primary demyelinating lesion. Brain 120 (Pt    8):1461-1483.-   12. Tang, L., T. P. Ugarova, E. F. Plow, and J. W. Eaton. 1996.    Molecular determinants of acute inflammatory responses to    biomaterials. J Clin Invest 97:1329-1334.-   13. Languino, L. R., J. Plescia, A. Duperray, A. A. Brian, E. F.    Plow, J. E. Geltosky, and D. C. Alfieri. 1993. Fibrinogen mediates    leukocyte adhesion to vascular endothelium through an    ICAM-1-dependent pathway. Cell 73:1423-1434.-   14. Herwald, H., H. Cramer, M. Morgelin, W. Russell, U.    Sollenberg, A. Norrby Teglund, H. Flodgaard, L. Lindbom, and L.    Bjorck. 2004. M Protein, a Classical Bacterial Virulence    Determinant, Forms Complexes with Fibrinogen that Induce Vascular    Leakage. Cell 116:367-379.-   15. Flick, M. J., X. Du, D. P. Witte, M. Jirouskova, D. A.    Soloviev, S. J. Busuttil, E. F. Plow, and J. L. Degen. 2004.    Leukocyte engagement of fibrin(ogen) via the integrin receptor    alphaMbeta2/Mac-1 is critical for host inflammatory response in    vivo. J Clin Invest 113:1596-1606.-   16. Adams, R. A., M. Passino, B. D. Sachs, T. Nuriel, and K.    Akassoglou. 2004. Fibrin mechanisms and functions in nervous system    pathology. Mol Intery 4:163-176.-   17. Flick, M. J., X. Du, and J. L. Degen. 2004. Fibrin(ogen)-alpha M    beta 2 interactions regulate leukocyte function and innate immunity    in vivo. Exp Biol Med (Maywood) 229:1105-1110.-   18. Akassoglou, K., R. A. Adams, J. Bauer, V. Tseveleki, P.    Mercado, H. Lassmann, L. Probed, and S. Strickland. 2004. Fibrin    depletion decreases inflammation and delays the onset of    demyelination in a tumor necrosis factor transgenic mouse model for    multiple sclerosis. Proc Natl Acad Sci USA 101:6698-6703.-   19. Paterson, P. Y. 1976. Experimental allergic encephalomyelitis:    role of fibrin deposition in immunopathogenesis of inflammation in    rats. Fed Proc 35:2428-2434. [0193]-   20. CoIler, B. S. 1997. Platelet GPIIb/IIIa antagonists: the first    anti-integrin receptor therapeutics. J Clin Invest 99:1467-1471.-   21. Rotshenker, S. 2003. Microglia and macrophage activation and the    regulation of complement-receptor-3 (CR3/MAC-1)-mediated myelin    phagocytosis in injury and disease. J Mol Neurosci 21:65-72.-   22. van der Laan, L. J., S. R. Ruuls, K. S. Weber, I. J.    Lodder, E. A. Dopp, and C. D. Dijkstra. 1996. Macrophage    phagocytosis of myelin in vitro determined by flow cytometry:    phagocytosis is mediated by CR3 and induces production of tumor    necrosis factor-alpha and nitric oxide. J Neuroimmuno170:145-152.-   23. Reichert, F., U. Slobodov, C. Makranz, and S. Rotshenker. 2001.    Modulation (inhibition and augmentation) of complement    receptor-3-mediated myelin phagocytosis. Neurobiol Dis 8:504-512.-   24. Alfieri, D. C., R. Bader, P. M. Mannucci, and T. S.    Edgington. 1988. Oligospecificity of the cellular adhesion receptor    Mac-1 encompasses an inducible recognition specificity for    fibrinogen. J Cell Biol 107:1893-1900.-   25. Lishko, V. K., B. Kudryk, V. P. Yakubenko, V. C. Yee, and T. P.    Ugarova. 2002. Regulated unmasking of the cryptic binding site for    integrin alpha M beta 2 in the gamma C-domain of fibrinogen.    Biochemistry 41:12942-12951.-   26. Fenteany, G., and M. Glogauer. 2004. Cytoskeletal remodeling in    leukocyte function. Curr Opin Hematol 11:15-24.-   27. Harrison, R. E., and S. Grinstein. 2002. Phagocytosis and the    microtubule cytoskeleton. Biochem Cell Biol 80:509-515.-   28. Ehlers, M. R. W. 2000. CR3: a general purpose adhesion    recognition receptor essential for innate immunity. Microbes and    Infection 2:289-294.-   29. Ugarova, T. P., D. A. Solovjov, L. Zhang, D. I. Loukinov, V. C.    Yee, L. V. Medved, and E. F. Plow. 1998. Identification of a novel    recognition sequence for integrin alphaM beta2 within the    gamma-chain of fibrinogen. J Biol Chem 273:22519-22527.-   30. Lehnardt, S., L. Massillon, P. Follett, F. E. Jensen, R.    Ratan, P. A. Rosenberg, J. J. Volpe, and T. Vartanian. 2003.    Activation of innate immunity in the CNS triggers neurodegeneration    through a Toll-like receptor 4-dependent pathway. Proc Natl Acad Sci    USA 100:8514-8519.-   31. Lehnardt, S., C. Lachance, S. Patrizi, S. Lefebvre, P. L.    Follett, F. E. Jensen, P. A. Rosenberg, J. J. Volpe, and T.    Vartanian. 2002. The toll-like receptor TLR4 is necessary for    lipopolysaccharide-induced oligodendrocyte injury in the CNS. J    Neurosci 22:2478-2486.-   32. Stephens, L., C. Ellson, and P. Hawkins. 2002. Roles of PI3Ks in    leukocyte chemotaxis and phagocytosis. Curr Opin Cell Biol    14:203-213.-   33. Caron, E., and A. Hall. 1998. Identification of two distinct    mechanisms of phagocytosis controlled by different Rho GTPases.    Science 282:1717-1721.-   34. Bell, W. R., S. S. Shapiro, J. Martinez, and H. L. Nossel. 1978.    The effects of ancrod, the coagulating enzyme from the venom of    Malayan pit viper (A. rhodostoma) on prothrombin and fibrinogen    metabolism and fibrinopeptide A release in man. J Lab Clin Med    91:592-604.-   35. Youssef, S., O. Stuve, J. C. Patarroyo, P. J. Ruiz, J. L.    Radosevich, E. M. Hur, M. Bravo, D. J. Mitchell, R. A. Sobel, L.    Steinman, and S. S. Zamvil. 2002. The HMG CoA reductase inhibitor,    atorvastatin, promotes a Th2 bias and reverses paralysis in central    nervous system autoimmune disease. Nature 420:78-84.-   36. Ugarova, T. P., and V. P. Yakubenko. 2001. Recognition of    fibrinogen by leukocyte integrins. Ann N Y Acad Sci 936:368-385.-   37. Ugarova, T. P., V. K. Lishko, N. P. Podolnikova, N.    Okumura, S. M. Merkulov, V. P. Yakubenko, V. C. Yee, S. T. Lord,    and T. A. Haas. 2003. Sequence gamma 377 395(P2), but not gamma    190-202(P1), is the binding site for the alpha MI-domain of integrin    alpha M beta 2 in the gamma C-domain of fibrinogen. Biochemistry    42:93659373.-   38. Ross, T. M., P. M. Martinez, J. C. Renner, R. G. Thorne, L. R.    Hanson, and W. H. Frey, 2nd. 2004. Intranasal administration of    interferon beta bypasses the blood-brain barrier to target the    central nervous system and cervical lymph nodes: a non-invasive    treatment strategy for multiple sclerosis. J Neuroimmunol 151:66-77.-   39. Yura, M., I. Takahashi, S. Terawaki, T. Hiroi, M. N. Kweon, Y.    Yuki, and H. Kiyono. 2001. Nasal administration of cholera toxin    (CT) suppresses clinical signs of experimental autoimmune    encephalomyelitis (EAE). Vaccine 20:134139.-   40. Xiao, B. G., X. F. Bai, G. X. Zhang, and H. Link. 1998.    Suppression of acute and protracted-relapsing experimental allergic    encephalomyelitis by nasal administration of low-dose IL-10 in rats.    J Neuroimmunol 84:230-237.-   41. Ishikawa, M., Y. Jin, H. Guo, H. Link, and B. G. Xiao. 1999.    Nasal administration of transforming growth factor-beta1 induces    dendritic cells and inhibits protracted relapsing experimental    allergic encephalomyelitis. Mult Scler 5:184-191.-   42. Tran, E. H., H. Hardin-Pouzet, G. Verge, and T. Owens. 1997.    Astrocytes and microglia express inducible nitric oxide synthase in    mice with experimental allergic encephalomyelitis. J    Neuroimmuno174:121-129.-   43. Brosnan, C. F., M. B. Bornstein, and B. R. Bloom. 1981. The    effects of macrophage depletion on the clinical and pathologic    expression of experimental allergic encephalomyelitis. J Immunol    126:614-620.-   44. Laudano, A. P., and R. F. Doolittle. 1981. Influence of calcium    ion on the binding of fibrin amino terminal peptides to fibrinogen.    Science 212:457-459.-   45. Vos, C. M., J. J. Geurts, L. Montagne, E. S. van Haastert, L.    Bo, P. van der Valk, F. Barkhof, and H. E. de Vries. 2005.    Blood-brain barrier alterations in both focal and diffuse    abnormalities on postmortem MRI in multiple sclerosis. Neurobiol Dis    20:953-960.-   46. Monje, M. L., H. Toda, and T. D. Palmer. 2003. Inflammatory    blockade restores adult hippocampal neurogenesis. Science    302:1760-1765.-   47. Inoue, A., C. S. Koh, K. Shimada, N. Yanagisawa, and K.    Yoshimura. 1996. Suppression of cell-transferred experimental    autoimmune encephalomyelitis in defibrinated Lewis rats. J    Neuroimmunol 71:131-137.-   48. Loike, J. D., B. Sodeik, L. Cao, S. Leucona, J. I. Weitz, P. A.    Detmers, S. D. Wright, and S. C. Silverstein. 1991. CD11c/CD18 on    neutrophils recognizes a domain at the N terminus of the A alpha    chain of fibrinogen. Proc Natl Acad Sci USA 88:1044-1048.-   49. McMahon, E. J., S. L. Bailey, C. V. Castenada, H. Waldner,    and S. D. Miller. 2005. Epitope spreading initiates in the CNS in    two mouse models of multiple sclerosis. Nat Med 11:335-339.-   50. Greter, M., F. L. Heppner, M. P. Lemos, B. M. Odermatt, N.    Goebels, T. Laufer, R. J. Noelle, and B. Becher. 2005. Dendritic    cells permit immune invasion of the CNS in an animal model of    multiple sclerosis. Nat Med 11:328-334.-   51. Carson, M. J. 2002. Microglia as liaisons between the immune and    central nervous systems: functional implications for multiple    sclerosis. Glia 40:218-231.-   52. Lucchinetti, C. F., W. Bruck, M. Rodriguez, and H.    Lassmann. 1996. Distinct patterns of multiple sclerosis pathology    indicates heterogeneity on pathogenesis. Brain Pathol 6:259-274.-   53. Barnett, M. H., A. P. Henderson, and J. W. Prineas. 2006. The    macrophage in MS: just a scavenger after all? Pathology and    pathogenesis of the acute MS lesion. Mult Scler 12:121-132.-   54. Noseworthy, J., D. Miller, and A. Compston. 2006.    Disease-modifying treatments in multiple sclerosis. McAlpine's    Multiple Sclerosis, Churchill Livingstone, Elsevier, Philadelphia,    Pa. 729-802 pp.-   55. Akassoglou, K., W.-M. Yu, P. Akpinar, and S. Strickland. 2002.    Fibrin inhibits peripheral nerve regeneration by arresting Schwann    cell differentiation. Neuron 33:861-875.-   56. Karnezis, T., W. Mandemakers, J. L. McQualter, B. Zheng, P. P.    Ho, K. A. Jordan, B. M. Murray, B. Barres, M. Tessier-Lavigne,    and C. C. Bernard. 2004. The neurite outgrowth inhibitor Nogo A is    involved in autoimmune-mediated demyelination. Nat Neurosci    7:736-744.-   57. Akassoglou, K., J. Bauer, G. Kassiotis, M. Pasparakis, H.    Lassmann, G. Kollias, and L. Probert. 1998. Oligodendrocyte    apoptosis and primary demyelination induced by local TNF/p55TNF    receptor signaling in the central nervous system of transgenic mice:    models for multiple sclerosis with primary oligodendrogliopathy. Am    J Pathol 153:801-813.-   58. Akassoglou, K., B. Malester, J. Xu, L. Tessarollo, J.    Rosenbluth, and M. V. Chao. 2004. Brain-specific deletion of    neuropathy target esterase/swisscheese results in neurodegeneration.    Proc Natl Acad Sci USA 101:5075-5080.-   59. Siao, C. J., S. R. Fernandez, and S. E. Tsirka. 2003. Cell    type-specific roles for tissue plasminogen activator released by    neurons or microglia after excitotoxic injury. J Neurosci    23:3234-3242.-   60. Seasholtz, T. M., J. Radeff-Huang, S. A. Sagi, R. Matteo, J. M.    Weems, A. S. Cohen, J. R. Feramisco, and J. H. Brown. 2004.    Rho-mediated cytoskeletal rearrangement in response to LPA is    functionally antagonized by Racl and PIP2. J Neurochem 91:501 512.

What is claimed is:
 1. A method for the prevention or treatment of adegenerative disorder of the nervous system in a subject in needcomprising administering to the subject an effective amount of acomposition comprising a pharmaceutically acceptable carrier and anagent that inhibits fibrin γ377-395 (SEQ ID NO:1) specific binding toMac-1.
 2. The method of claim 1, wherein the agent is an antibody thatspecifically binds to a peptide consisting essentially of an amino acidsequence at least 89% identical to SEQ ID NO:1.
 3. The method of claim2, wherein the antibody is a monoclonal antibody, or an antigen-bindingfragment selected from the group consisting of Fab, Fab′, F(ab′)₂ andF(v).
 4. The method of claim 2, wherein the antibody is a chimeric,humanized, or human antibody.
 5. The method of claim 1, wherein thedegenerative disorder of the nervous system involves leakage offibrinogen perivascularly in the brain.
 6. The method of claim 5,wherein the degenerative disorder of the nervous system is selected fromthe group consisting of multiple sclerosis, spinal cord injury, strokeand Alzheimer's Disease.
 7. The method of claim 2, wherein the aminoacid sequence is at least 94% identical to SEQ ID NO:1.
 8. The method ofclaim 2, wherein the amino acid sequence is shown in SEQ ID NO:1.
 9. Amethod of inhibiting fibrin binding to Mac-1 in a subject in needcomprising administering to the subject an effective amount of acomposition comprising an agent that inhibits fibrin γ377-395 (SEQ IDNO:1) specific binding to Mac-1.
 10. The method of claim 9, wherein themethod inhibits fibrin mediated microglia activation.
 11. The method ofclaim 9, wherein the method is for the prevention or treatment of adegenerative disorder of the nervous system.
 12. The method of claim 9,wherein the agent is an antibody that specifically binds to a peptideconsisting essentially of an amino acid sequence at least 89% identicalto SEQ ID NO:1.
 13. The method of claim 12, wherein the amino acidsequence is at least 94% identical to SEQ ID NO:1.
 14. The method ofclaim 12, wherein the amino acid sequence is shown in SEQ ID NO:1. 15.The method of claim 12, wherein the antibody is a chimeric, humanized,or human antibody.